Biopolymer-based inks and use thereof

ABSTRACT

The present application discloses biopolymer-based ink formulations that are useful for inkjet printing and other applications. Related methods are also disclosed.

RELATED APPLICATIONS

This continuation application claims the benefit to U.S. applicationSer. No. 14/647,470 filed May 27, 2015, now U.S. Pat. No. 10,035,920;which is a National Stage entry of PCT/US2013/0724435 filed Nov. 27,2013 which claims benefit of and priority to U.S. provisionalapplications 61/730,453 filed Nov. 27, 2012, entitled “SILK FIBROINPROTEIN INKS” and 61/826,458 filed May 22, 2013, also entitled “SILKFIBROIN PROTEIN INKS,” the entire contents of each of which areincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grantsFA9550-14-1-0015 awarded by the United States Air Force andN00014-13-1-0596 awarded by the United States Navy. The government hascertain rights in the invention.

BACKGROUND

Inkjet printing is a type of computer-based printing that creates adigital image by propelling droplets of ink onto a substrate, typicallypaper. The concept of inkjet printing has been around for over a centurybut was made readily accessible to consumers in the latter half of the20th century. Inkjet printing can be also employed for direct materialdeposition, which is an emerging manufacturing technique today.

The field of computer-assisted printing has seen vast technologicaladvancement in recent years, including the 3D printing technology usedto fabricate computer-designed objects by so-called “additivemanufacturing.” Typically, the process of 3D printing involves serialprinting (i.e., layering) of particles or 3D dots comprised ofmaterials, such as thermoplastics, metal alloys, and plasters.

The inkjet technology as applied to biological materials, however,generally faces a number of technical obstacles.

SUMMARY OF THE INVENTION

Among other things, the present application provides the use of certainbiopolymers for preparing printable liquids, i.e., “biopolymer-basedinks” or “bio-inks.” In particular, the present invention encompassesthe recognition that certain polypeptides have material and chemicalfeatures that are suitable to be formulated into such inks.

More specifically, the present invention includes the finding thatcertain polypeptides of structural proteins in origin are especiallysuited for fabricating micro- and nano-scale (i.e., sub-micron) printedstructures. Such structures include two-dimensional (2D) structures andthree-dimensional (3D) structures.

Furthermore, unique material features of biopolymer-based inks allowincorporation of a variety of additives (e.g., agents or dopants) forfunctionalization, which can be stabilized within the ink. In someembodiments, provided bio-ink compositions may further contain otheradditives, such as excipients, chelating agents, defoamers, etc., amongothers.

The invention is useful for a wide range of applications, including butnot limited to, optoelectonics, photonics, therapeutics, tissueengineering such as intelligent implants, synthetic biology, and avariety of consumer products.

Accordingly, in one aspect, the invention provides aqueous (i.e.,water-based) biopolymer inks. In some embodiments, described biopolymerinks comprise a structural protein. In some embodiments, describedbiopolymer inks comprise a structural protein having a specified rangeor ranges of molecular weights (e.g., fragments). In some embodiments,the specified range includes between about 3.5 kDa and 120 kDa. In someembodiments, described biopolymer inks are substantially free of proteinfragments exceeding a specified molecular weight. For example, in someembodiments, described biopolymer inks are substantially free of proteinfragments over 200 kDa. “Substantially free” means that it is absent orpresent at a concentration below detection measured by any art-acceptedmeans, such that it is considered negligible.

According to the invention, described biopolymer inks have a viscosityofbetween about 1-20 centipoise (cP), where 1 cP=1 mPa·s=0.001 Pa·s, asmeasured at room temperature of between about 18-26° C. In someembodiments, described biopolymer inks further comprise one or moresuitable viscosity-modifying agents (i.e., viscosity modifiers orviscosity adjusters).

Biopolymer ink compositions in accordance with the present invention mayalso contain one or more added agents, or additives, such as dopants. Insome embodiments, such added agents are stabilized by the inkcomposition. In some embodiments, such added agents are stabilized bythe biopolymer (e.g., structural protein) present in the inkcomposition.

In another aspect, the present invention provides a small volume unit ofan aqueous composition comprising a low molecular weight structuralprotein. Such a unit is an liquid droplet of between about 0.1-100 pL.In some embodiments, such an aqueous unit composition contains the lowmolecular weight structural protein at a concentration of about0.1-10.0%. In some embodiments, such an aqueous unit composition has aviscosity of between about 1-20 centipoise or 1-20 mPa·s.

In a further aspect, the invention provides an array of printed units,which may be a semi-solid or solid form.

According to the invention, a printed array may comprise a substrate,upon which a plurality of dot units is deposited, wherein each dot unitcomprises a low molecular weight structural protein. Each dot unit istypically between about 0.1-250 μm in diameter. Such dot units may bedeposited upon the substrate in a predetermined spatial pattern,including regular and irregular patterns. Printed structures asdescribed herein may be a 2D or a 3D structure.

In some embodiments, described printed arrays have a resolution ofbetween about 50-20,000 dpi, depending on a variety of parameters asfurther described herein.

In any of the embodiments, such printed structures, which are made ofdot units as described, may be deposited on a suitable substrate.

In yet a further aspect, the invention provides methods for printing astructure. The described methods involve providing a protein-based inkcomprising a low molecular weight structural protein of suitablecharacteristics and depositing the protein-based ink onto a substrate ina predetermined spatial pattern. According to the invention, each liquiddroplet has a volume of between about 0.1-100 pL.

The invention also includes methods for manufacturing biopolymer-basedink compositions (i.e., “bio-inks”). In some embodiments, providedmanufacturing methods include providing an aqueous solution comprising alow molecular weight structural protein (or fragment thereof), andconfirming or adjusting the aqueous solution so as to achieve a suitableparameter(s), such as viscosity, surface tension, density (specificgravity), pH, etc.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 shows a summary of silk fibroin stabilization effects onimmobilized compounds.

FIG. 2 shows materials formed with regenerated silk fibroin.

FIG. 3 shows a protocol to obtain printable silk inks from raw cocoon.

FIGS. 4A-4D shows inkjet printing of biopolymer inks on differentsubstrates.

FIG. 5 shows an overview of biopolymer ink design, device design, andbiopolymer ink-substrate interaction.

FIG. 6 shows an inkjet materials printer.

FIG. 7 shows a schematic of components of the inkjet printer of FIG. 6.

FIG. 8 shows scanning electron microscope (SEM) images of inkjet-printedsilver nanoparticle ink before and after sintering at 100 degrees C. for15 minutes.

FIG. 9 shows an inkjet printed RFID tag.

FIG. 10 illustrates simulated and measured intensity over frequency ofthe inkjet-printed RFID tag of FIG. 9.

FIG. 11 shows an SEM image of human fibrosarcoma cells after inkjetprinting.

FIG. 12 shows patterns for a pattern editor.

FIG. 13 shows patterns for a pattern editor.

FIG. 14 shows a cartridge settings interface with voltage settings.

FIG. 15 shows a cartridge tab of the cartridge settings interface ofFIG. 14.

FIG. 16 shows a cleaning cycles tab of the cartridge settings interfaceof FIG. 14.

FIG. 17 shows a piezo actuated nozzle unit of an inkjet printer when awaveform begins.

FIG. 18 shows the piezo actuated nozzle unit of FIG. 17 in a firstwaveform phase.

FIG. 19 shows the piezo actuated nozzle unit of FIG. 17 in a secondwaveform phase.

FIG. 20 shows the piezo actuated nozzle unit of FIG. 17 in a thirdwaveform phase.

FIG. 21 shows biopolymer lines printed under different voltage: 1) 15 vvoltage, 65 um; 2) 20 v voltage, 100 um; and 3) 25 v voltage, 110 um.

FIG. 22 shows a waveform for biopolymer ink printing.

FIG. 23 shows a one-nozzle printing with a 20 μm line width.

FIG. 24 shows a multi-nozzle printing with a 240 μm line width.

FIG. 25 shows biopolymer drops.

FIG. 26 shows biopolymer dots printed on a silicon wafer.

FIG. 27 shows biopolymer dots printed on acrylic.

FIG. 28 shows a one-layer biopolymer pattern on a silicon wafer.

FIG. 29 shows one-layer lines.

FIG. 30 shows a three-layer biopolymer pattern on a silicon wafer.

FIG. 31 shows three-layer lines.

FIG. 32 shows a twenty-layer biopolymer pattern on a silicon wafer.

FIG. 33 shows a cross biopolymer line pattern.

FIG. 34 shows a cross biopolymer line pattern with capillaryinstability.

FIG. 35 shows a cross silk line patter with capillary instability, withan enlarged inset view.

FIG. 36 shows one-layer 2D biopolymer patterns.

FIG. 37 shows one-layer 2D patterns showing diffraction gratingpatterns.

FIG. 38 shows multi-layer 2D patterns.

FIG. 39 shows an enlarged partial view of the multi-layer 2D pattern ofFIG. 38.

FIG. 40 shows a) a silk pattern before annealing and b) the silk patternafter annealing.

FIG. 41 shows printing layers vs. thickness of food color biopolymerpatterns.

FIG. 42 shows printing layers vs. thickness of high refractive indexbiopolymer patterns.

FIG. 43 shows printing layers vs. thickness of biopolymer patterns.

FIG. 44 shows a comparison of printing layers vs. thickness of differentsilk inks.

FIG. 45 shows one-layer biopolymer patterns on acrylic.

FIG. 46 shows gold nanoparticle doped silk ink in a tube.

FIG. 47 shows a gold nanoparticle doped silk dot pattern on paper.

FIG. 48 shows an infrared view of the pattern of FIG. 47 when exposed togreen light radiation.

FIG. 49 shows printed HRP doped biopolymer changing color to blue whensprayed with a tetramehtylbenzidine (TMB) solution.

FIG. 50 shows two clean bacterial inhibition zones in a bacterial growthpetri dish.

FIG. 51 shows a bacterial growth inhibition zone in the shape of anarrow.

FIG. 52 shows a single color biopolymer pattern on silk textile.

FIG. 53 shows a single color biopolymer pattern on silk textile afterdry cleaning.

FIG. 54 shows multiple-color silk pattern on silk textile before andafter alignment optimization.

FIG. 55 shows cashmere (keratin) ink in the form of dots on a siliconsubstrate.

FIG. 56 shows cashmere (keratin) ink in the form of a line on a siliconsubstrate.

FIG. 57 shows cashmere (keratin) ink in the form of lines on a siliconsubstrate.

FIG. 58 shows cashmere (keratin) ink in the form of dots on a glasssubstrate.

FIG. 59 shows cashmere (keratin) ink in the form of lines on a glasssubstrate.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Inkjet Printing Technology

Inkjet printing (IJP) is an easy, inexpensive and widely accessibletechnology spread around the world for several decades. The fortune ofIJP is tied to the pervasiveness of personal computing, as for the lasttwo decades it has represented one of the fundamental accessories forany PC workstation.

UP is based on the use of electrical actuators to eject picoliter (pL)volumes of liquid from micrometer-wide nozzles onto a substrate in adefined pattern. IJP has gained extensive acceptance in microfabricationfor basic patterning and rapid fabrication. While the most popularpurpose of IJP technology remains printing paper documents, it has alsobeen applied in organic electronics, chemical synthesis, sensorfabrication, combinatorial chemistry and biology.

Inkjet printing can be divided into two categories: (1) drop-on-demand(DoD) or impulse inkjet, where droplets are generated when required; and(2) continuous inkjet, in which droplets are deflected from a continuousstream to a substrate when needed.

Inkjet printing can be further subdivided according to the specificmeans of generating droplets, such as piezoelectric, thermal andelectrostatic. Each of these techniques has specific ranges of operationthat limit their applicability. Such variables include: operatingtemperature range, material throughput, reproducibility of droplets,precision of deposition, range of printable viscosities, range of shearforces within the nozzle, reservoir volume and the number of fluids thatmay be printed during at the same time. Droplet size involves,typically, volumes ranging from 1.5 pL to 5 nL at a rate of 0-25 kHz fordrop-on-demand printers (and up to 1 MHz for continuous printheads).

Alternative Printing Methods

Electrohydrodynamic jet printing (EHJP) can produce features as small as1 μm wide lines, which is typically an order of magnitude smaller thaninkjet printing. Naturally, the droplets produced by this technique arealso smaller, being in the femto-liter region. Such small droplet sizesare of interest since this means that less material can be dispensedwith more spatial control, which couples with the ongoingminiaturization seen in many applications. An open question to beaddressed is whether the EHJP droplet ejection method affects thematerial contained within the ink. Whereas inkjet printers eject theirdroplets from within the nozzle, EHJ printers eject their droplets fromoutside the nozzle. The ink in an EHJ printer forms a droplet that isattached to the nozzle. This dome of ink is charged by a wire containedwithin the nozzle using voltages up to 200 V, which is necessary toovercome the surface tension and causes a Taylor cone to form. Thedroplets are ejected from the tip of the cone. This process likely makesprotein susceptible to electrical breakdown and droplet deflectionduring application of inks to substrates. Furthermore, the EHJP processis still in its infancy and it has not yet been applied to the fullrange of applications that inkjet printing has. In addition, EHJP isbased on electrostatic forces; meaning that the substrate must beconductive, which is also a limitation. Finally, the cost of thetechnique is another factor to consider.

Bio-Printing

Bio-printing is defined as the process of inkjet printing biomaterials.The field of bio-printing originated in the mid-1990s, with an expansionafter the turn in the new millennium. Several factors contributed tothis increase: by 1985 functional materials for sensing biologicalmatter (e.g. for glucose and urea) were spatially disposed to formmulti-analyte arrays. In 1987 the first patent for an inkjet-printedenzyme-based biosensor was filed. The robustness and versatility ofinkjet printing enabled researchers to modify the technique to meettheir needs. In particular, the mild conditions afforded by the UPprocess make it particularly suited for handling biological materials.Minimal sample contamination and waste together with the accuratecontrol and placement of pre-determined quantities of material are alsohighly appealing features. The possibility to integrate biomaterialswith IJP technology is convenient because it combines ease, low-cost androbustness. In particular, the use of UP to deposit proteins is ofparticular appeal given that the impossibility to apply conventionalpolymer processing techniques to proteins is one of the major hindranceto their applications as biomaterials.

Limitations to Protein Printing

During the course of research that led to the invention disclosedherein, several fundamental issues and technical obstacles related tothe methods employed were addressed. While inkjet printing of proteinshas been employed successfully with certain materials, it is not withoutlimitations. In particular, shear stress-induced denaturation andnon-specific protein adsorption on the inner surfaces of the printerhave been previously reported as problems to be addressed during theprocess set up. Prior to dealing with any protein-specificconsiderations, the first and foremost limitation to drop-on-demandinkjet printing of any material is its feasibility: whether or not thematerial can be formulated into an ink that is printable (i.e., stable,repeatable droplets are able to be ejected from the nozzle, with uniformvelocities and volumes). Generally, some of the most important intrinsicphysical properties determining printability of given ink include:viscosity (η), density (ρ), surface tension (γ), and nozzle diameter(α). Using a modified form of the Navier-Stokes equation, originallyproposed by Fromm, a dimensionless number Z based on the aforementionedphysical properties can be used to estimate whether or not the rightbalance between the capillary force, inertial force, and viscous forcemay be achieved for stable droplet formation by the following equation:

$Z = \frac{{\alpha\rho\gamma}^{1\text{/}2}}{\eta}$The mathematics involved in describing the theoretical printability offluids based on their calculated Z number has been outlined elsewhere ingreater detail by other authors, and in this review, it is sufficient tosay that a solution is theoretically printable when 1<Z<10. In mostinstances, any protein solution under consideration for printing may bedilute and aqueous, and thus have a density that is alreadypre-determined; for a given nozzle, the printability of a given proteinsolution is strongly affected at least by surface tension and viscosity.Surface Tension

Proteins formulated as bio-inks are inkjet-printed in aqueous solutions.The composition of the solution influences its surface tension. For highvalues of surface tension, the applied force that stretches andeventually causes the ejection of the drop is lower than the cohesivecounter-force. Indeed, the droplet resists to the external force,resulting in the lack of ejection.

Viscosity

Proteins are by nature macromolecules, and consequently the viscosity oftheir solutions is often dramatically affected by changes inconcentration. At higher concentrations, the capillary force isinsufficient to break the filament of the droplet during the ejection,and the droplet retracts back into the nozzle. For polymers, themicro-rheological explanation for this behavior is that the coiled andfolded polymer chains are elongated in the direction of flow into astretched state, which is accompanied by a strong increase of thehydrodynamic drag. However, most proteins, unlike synthetic linearpolymers, are not randomly coiled chains; rather, most proteins tend tobe carefully folded into organized structures in their native state, andthe degree to which proteins are either globular or fibrous plays animportant role in their intrinsic viscosity, which, consequently affectsthe maximum concentration of a printable solution. This implies arelative facility in the printing of globular protein such as enzymes,messenger/signaling, and transport proteins, while structural fibrousprotein, such as keratin, collagen, and elastin are impossible to printat relevant concentrations or in mild conditions (e.g., neutral pH,aqueous solution). This has a significant impact on the concentrationlimitation of printable solutions of specific categories of importantproteins, e.g., structural proteins. As an example, globular proteinsmay be easily printable in concentrations of 10 wt % or more with commondampened nozzles, but type I collagen solutions, for example, inconcentrations even as low as 0.3-0.5 wt % (a range commonly used forbiomedical applications) are unprintable with the same devices. While acommon technique for improving the printability of viscous inks is toraise the printing temperature, there are practical restrictions whichfurther limit the printability of structural proteins.

Bioprinting processes involve shear rates in the range of 2×10⁴ to 2×10⁶s⁻¹; while such shear rates pose no foreseeable problems for small,globular proteins, they are sufficiently high to compromise thestructural integrity of some of their more fragile, larger, counterparts(e.g. structural proteins). Another hindrance in bioprinting structuralproteins is the high compression rates used to generate droplets, whichmay result in the loss of both structural and biological properties,particularly in the absence of stabilizing additives.

Indeed, the highly organized nature of structural protein (collagen,elastin, keratin, fibrin, etc.) results in high surface tension,viscosity and shear stress, which are some of the main hindrance to thebioprinting process. This aspect is inherited from the tendency ofcell-produced monomers in more organized structures to self-assemblewhen exposed to physiological conditions. Such proteins are engineeredby Nature to self-organize in the extracellular space and not in anextrusion process upon exposure specific stimuli.

Composition of Novel, Printable Bio-Inks

Among other things, the present disclosure provides novel, water-based,biopolymer ink compositions (“bio-inks”) suitable for high resolutioninkjet printing. The techniques described herein opens a door to a newapproach of additive printing that enables the fabrication ofbiocompatible sub-micron and micro-scale structures with good precisionand reproducibility.

Non-limiting, exemplary ink formulations as a vehicle, suitable forcarrying out various embodiments of the present invention include thefollowing components: water (˜60-90%); water-soluble solvent such ashumectants for viscosity control (˜5-30%); dye or pigments (colorants)(˜1-10%); surfactant (˜0.1-10%); buffering agent (˜0.1-0.5%); and, otheradditives (˜1%), each of which is measured by weight.

Typically, an aqueous bio-ink composition in accordance with the presentinvention comprises the following three components: (i) a structuralprotein, (ii) a viscosity-modifying agent (i.e., viscosity modifier orviscosity adjuster), such as an amphiphilic agent, and (iii) water.

Structural Proteins

The present disclosure encompasses the recognition that it is possibleto control certain parameters of an aqueous biopolymer composition,thereby making it possible to be prepared and used as a liquid inkcomposition that can be readily printed on a substrate. In accordancewith the invention, upon disposition or printing, such ink compositionscan then form semi-solid or solid forms, which allows the fabrication ofeven sub-micron structures.

In some embodiments, a structural protein (such as silk fibroin andkeratin) is present in a bio-ink composition at a final concentration ofabout 0.1-10% by weight, e.g., about 0.1%, about 0.2%, about 0.3%, about0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, or greater.

In some embodiments, structural proteins (e.g., families and subfamiliesof such proteins) suitable for carrying out the present inventioninclude the following: fibroins, actins, collagens, catenins, claudins,coilins, elastins, elaunins, extensins, fibrillins, lamins, laminins,keratins, tublins, viral structural proteins, zein proteins (seedstorage protein) and any combinations thereof.

In some embodiments, it is particularly advantageous to use or selectprotein fragments of certain molecular weight ranges. The inventors ofthe present invention have determined that bio-inks made from astructural protein of molecular weights ranging between about 3.5 kDaand about 120 kDa are particularly useful. In some embodiments,therefore, provided bio-inks of the invention predominantly containstructural protein fragments ranging between about 3.5 kDa and about 120kDa, e.g., about 3.5 kDa and about 100 kDa, about 5 kDa and about 100kDa, about

Where such fragments correspond to reduced size, relative to thenaturally occurring full-length counterpart, such polypeptide fragmentsare broadly herein referred to as “low molecular weight” protein.

In some embodiments, polypeptide fragments corresponding to at least aportion of any one of the structural proteins listed above may be usedto make a bio-ink described herein. Such polypeptides suitable forpracticing the present invention may be produced from various sources,including a regenerated (e.g., purified) protein from natural sources,recombinant proteins produced in heterologous systems, synthetic orchemically produced peptides, or combination of these.

In some embodiments, described bio-inks may be prepared from apolypeptide corresponding to any one of the list provided above, with orwithout one or more sequence variations, as compared to the native orwild type counterpart. For example, in some embodiments, such variantsmay show at least 85% overall sequence identity as compared to a wildtype sequence, e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% overall sequence identity.

In some embodiments, bio-ink compositions of the present inventioncomprise silk fibroin, keratin, or combination thereof.

Silk Fibroin

Interestingly, silk fibroin has a different nature, being extruded froma living organism and changing its structure from globular to highlycrystalline during such process. The scope of this work thereforeincluded mimicking the natural silk fibroin extrusion process by inkjetprinting regenerated silk solution, pioneering a new way to process thisancient material and providing unprecedented functions to fibroin-basedbiomaterials.

Silk fibroin-based solutions may be formulated as “silk inks” for use inprinting. Accordingly, the invention includes silk fibroin-based inkcompositions and methods for manufacturing the same.

In some embodiments where silk fibroin polypeptides are used to preparepeptide-based aerogels in accordance with the present invention, suchsilk fibroin polypeptide may be a low molecular weight silk fibroinpolypeptide, ranging between about 3.5 kDa and about 120 kDa. Lowmolecular weight silk fibroin is described in detail in U.S. provisionalapplication 61/883,732, entitled “LOW MOLECULAR WEIGHT SILK FIBROIN ANDUSES THEREOF,” the entire contents of which are incorporated herein byreference.

In particular, such silk inks are suitable for use in conjunction withcommercially available inkjet printers. As described herein, in arelatively inexpensive, streamlined fashion, inkjet printing can beemployed for the fabrication of a wide range of nanostructures, usingsilk inks as a medium. These include two-dimensional (2D) andthree-dimensional (3D) nanostructures. Unique material features of silkfibroin allow incorporation of a variety of agents or dopants forfunctionalization, which are stabilized within the silk ink. Theinvention is applicable to an array of technologies, including but arenot limited to, optoelectonic, photonics, and therapeutics.

As used herein, the term “silk fibroin” useful for carrying out thepresent invention includes silkworm fibroin and insect or spider silkprotein. See e.g., Lucas et al., 13 Adv. Protein Chem. 107 (1958). Forexample, silk fibroin useful for the present invention may be thatproduced by a number of species, including, without limitation:Antheraea mylitta; Antheraea pernyi; Antheraea yamamai; Galleriamellonella; Bombyx mori; Bombyx mandarina; Galleria mellonella; Nephilaclavipes; Nephila senegalensis; Gasteracantha mammosa; Argiope aurantia;Araneus diadematus; Latrodectus geometricus; Araneus bicentenarius;Tetragnatha versicolor; Araneus ventricosus; Dolomedes tenebrosus;Euagrus chisoseus; Plectreurys tristis; Argiope trifasciata; and Nephilamadagascariensis.

In general, silk for use in accordance with the present invention may beproduced by any such organism, or may be prepared through an artificialprocess, for example, involving genetic engineering of cells ororganisms to produce a recombinant silk fibroin polypeptide and/orchemical synthesis. In some embodiments of the present invention, silkis produced by the silkworm, Bombyx mori.

As is known in the art, silks are modular in design, with large internalrepeats flanked by shorter (˜100 amino acid) terminal domains (N and Ctermini). Naturally occurring silk fibroin polypeptides have highmolecular weight (200 to 350 kDa or higher) with transcripts of 10,000base pairs and higher and >3000 amino acids (reviewed in Omenatto andKaplan (2010) Science 329: 528-531). The larger modular domains areinterrupted with relatively short spacers with hydrophobic charge groupsin the case of silkworm silk. N- and C-termini are involved in theassembly and processing of silks, including pH control of assembly. TheN- and C-termini are highly conserved, in spite of their relativelysmall size compared with the internal modules. An exemplary list ofsilk-producing species and corresponding silk proteins may be found inInternational Patent Publication Number WO 2011/130335, the entirecontents of which are incorporated herein by reference.

Cocoon silk produced by the silkworm, Bombyx mori, is of particularinterest because it offers low-cost, bulk-scale production suitable fora number of commercial applications, such as textile. Silkworm cocoonsilk contains two structural proteins, the fibroin heavy chain (˜350 kDa) and the fibroin light chain (˜25 k Da), which are associated with afamily of nonstructural proteins termed sericin, which glue the fibroinbrings together in forming the cocoon. The heavy and light chains offibroin are linked by a disulfide bond at the C-terminus of the twosubunits (Takei, F., Kikuchi, Y., Kikuchi, A., Mizuno, S. and Shimura,K. (1987) J. Cell Biol., 105, 175-180; Tanaka, K., Mori, K. and Mizuno,S. (1993) J. Biochem. (Tokyo), 114, 1-4; Tanaka, K., Kajiyama, N.,Ishikura, K., Waga, S., Kikuchi, A., Ohtomo, K., Takagi, T. and Mizuno,S. (1999) Biochim. Biophys. Acta, 1432, 92-103; Y Kikuchi, K Mori, SSuzuki, K Yamaguchi and S Mizuno, Structure of the Bombyx mori fibroinlight-chain-encoding gene: upstream sequence elements common to thelight and heavy chain, Gene 110 (1992), pp. 151-158). The sericins are ahigh molecular weight, soluble glycoprotein constituent of silk whichgives the stickiness to the material. These glycoproteins arehydrophilic and can be easily removed from cocoons by boiling in water.This process is often referred to as “degumming.”

As used herein, the term “silk fibroin” embraces silk fibroin protein,whether produced by silkworm, spider, or other insect, or otherwisegenerated (Lucas et al., Adv. Protein Chem., 13: 107-242 (1958)). Insome embodiments, silk fibroin is obtained from a solution containing adissolved silkworm silk or spider silk. For example, in someembodiments, silkworm silk fibroins are obtained, from the cocoon ofBombyx mori. In some embodiments, spider silk fibroins are obtained, forexample, from Nephila clavipes. In the alternative, in some embodiments,silk fibroins suitable for use in the invention are obtained from asolution containing a genetically engineered silk harvested frombacteria, yeast, mammalian cells, transgenic animals or transgenicplants. See, e.g., WO 97/08315 and U.S. Pat. No. 5,245,012, each ofwhich is incorporated herein as reference in its entirety.

Thus, in some embodiments, a silk solution is used to fabricatecompositions of the present invention contain fibroin proteins,essentially free of sericins. Provided silk fibroin particlescontemplated herein are essentially free of sericins, unless otherwiseexplicitly specified. “Essentially free of sericins” means that suchcompositions contain no (e.g., undetectable) or little (i.e., traceamount) sericin such that one of ordinary skill in the pertinent artwill consider negligible for a particular use.

In some embodiments, silk solutions used to fabricate variouscompositions of the present invention contain the heavy chain offibroin, but are essentially free of other proteins. In otherembodiments, silk solutions used to fabricate various compositions ofthe present invention contain both the heavy and light chains offibroin, but are essentially free of other proteins. In certainembodiments, silk solutions used to fabricate various compositions ofthe present invention comprise both a heavy and a light chain of silkfibroin; in some such embodiments, the heavy chain and the light chainof silk fibroin are linked via at least one disulfide bond. In someembodiments where the heavy and light chains of fibroin are present,they are linked via one, two, three or more disulfide bonds.

Although different species of silk-producing organisms, and differenttypes of silk, have different amino acid compositions, various fibroinproteins share certain structural features. A general trend in silkfibroin structure is a sequence of amino acids that is characterized byusually alternating glycine and alanine, or alanine alone. Suchconfiguration allows fibroin molecules to self-assemble into abeta-sheet conformation. These “Ala-rich” hydrophobic blocks aretypically separated by segments of amino acids with bulky side-groups(e.g., hydrophilic spacers).

In some embodiments, core repeat sequences of the hydrophobic blocks offibroin are represented by the following amino acid sequences and/orformulae:

(SEQ ID NO: 1) (GAGAGS)₅₋₁₅; (SEQ ID NO: 2) (GX)₅₋₁₅ (X = V, I, A); (SEQID NO: 3) GAAS; (SEQ ID NO: 4) (S₁₋₂A₁₁₋₁₃); (SEQ ID NO: 5) GX₁₋₄ GGX;(SEQ ID NO: 6) GGGX (X = A, S, Y, R, D, V, W, R, D); (SEQ ID NO: 7)(S1-2A1-4)₁₋₂; (SEQ ID NO: 8) GLGGLG; (SEQ ID NO: 9) GXGGXG (X = L, I,V, P); (SEQ ID NO: 10) GPX (X = L, Y, I); (GP(GGX)₁₋₄ Y)n (X = Y, V, S,A); (SEQ ID NO: 11) GRGGAn; (SEQ ID NO: 12) GGXn (X = A, T, V, S);GAG(A)₆₋₇GGA; and (SEQ ID NO: 13) GGX GX GXX (X = Q, Y, L, A, S, R).

In some embodiments, a fibroin peptide contains multiple hydrophobicblocks, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 and 20 hydrophobic blocks within the peptide. In some embodiments, afibroin peptide contains between 4-17 hydrophobic blocks. In someembodiments of the invention, a fibroin peptide comprises at least onehydrophilic spacer sequence (“hydrophilic block”) that is about 4-50amino acids in length. Non-limiting examples of the hydrophilic spacersequences include:

(SEQ ID NO: 14) TGSSGFGPYVNGGYSG; (SEQ ID NO: 15) YEYAWSSE; (SEQ ID NO:16) SDFGTGS; (SEQ ID NO: 17) RRAGYDR; (SEQ ID NO: 18) EVIVIDDR; (SEQ IDNO: 19) TTIIEDLDITIDGADGPI and (SEQ ID NO: 20) TISEELTI.

In certain embodiments, a fibroin peptide contains a hydrophilic spacersequence that is a derivative of any one of the representative spacersequences listed above. Such derivatives are at least 75%, at least 80%,at least 85%, at least 90%, or at least 95% identical to any one of thehydrophilic spacer sequences.

In some embodiments, a fibroin peptide suitable for the presentinvention contains no spacer.

As noted, silks are fibrous proteins and are characterized by modularunits linked together to form high molecular weight, highly repetitiveproteins. These modular units or domains, each with specific amino acidsequences and chemistries, are thought to provide specific functions.For example, sequence motifs such as poly-alanine (polyA) andpolyalanine-glycine (poly-AG) are inclined to be beta-sheet-forming; GXXmotifs contribute to 31-helix formation; GXG motifs provide stiffness;and, GPGXX (SEQ ID NO: 22) contributes to beta-spiral formation. Theseare examples of key components in various silk structures whosepositioning and arrangement are intimately tied with the end materialproperties of silk-based materials (reviewed in Omenetto and Kaplan(2010) Science 329: 528-531).

It has been observed that the beta-sheets of fibroin proteins stack toform crystals, whereas the other segments form amorphous domains. It isthe interplay between the hard crystalline segments, and the strainedelastic semi amorphous regions, that gives silk its extraordinaryproperties. Non-limiting examples of repeat sequences and spacersequences from various silk-producing species are provided in Anexemplary list of hydrophobic and hydrophilic components of fibroinsequences may be found in International Patent Publication Number WO2011/130335, the entire contents of which are incorporated herein byreference.

In any of the embodiments contemplated herein, silk fibroin polypeptidesof various molecular weights (e.g., fragments) may be used. In someembodiments, for example, provided silk fibroin hydrogel comprises silkfibroin polypeptides having an average molecular weight of between about3.5 kDa and about 350 kDa. Non-limiting examples of suitable ranges ofsilk fibroin fragments include, but are not limited to: silk fibroinpolypeptides have an average molecular weight of between about 3.5 kDaand about 200 kDa; silk fibroin polypeptides have an average molecularweight of between about 3.5 kDa and about 200 kDa; silk fibroinpolypeptides have an average molecular weight of between about 3.5 kDaand about 120 kDa; silk fibroin polypeptides have an average molecularweight of between about 25 kDa and about 200 kDa, and so on. Silkfibroin polypeptides that are “reduced” in size, for instance, smallerthan the original or wild type counterpart, may be referred to as “lowmolecular weight silk fibroin.”

In some embodiments, provided silk fibroin particles are prepared fromcomposition comprising a population of silk fibroin fragments having arange of molecular weights, characterized in that: no more than 15% oftotal weight of the silk fibroin fragments in the population has amolecular weight exceeding 200 kDa, and at least 50% of the total weightof the silk fibroin fragments in the population has a molecular weightwithin a specified range, wherein the specified range is between about3.5 kDa and about 120 kDa.

For more details related to low molecular weight silk fibroins, see:U.S. provisional application 61/883,732, entitled “LOW MOLECULAR WEIGHTSILK FIBROIN AND USES THEREOF,” the entire contents of which areincorporated herein by reference.

Keratins

Keratin is a large family of fibrous structural proteins. Keratin is thekey structural material making up the outer layer of human skin. It isalso the key structural component of hair and nails. Keratin monomersassemble into bundles to form intermediate filaments, which are toughand insoluble and form strong unmineralized tissues found in reptiles,birds, amphibians, and mammals. The only other biological matter knownto approximate the toughness of keratinized tissue is chitin.

Viscosity-Modifying Agents

As stated, biopolymer-based ink formulations described herein typicallycontain at least one viscosity-modifying agent, also referred to asviscosity modifiers or viscosity adjusters. As described in detailherein, having the optimal range of viscosity is important for ensuinghigh quality, reproducible inkjet printing. As such, one or more of anysuitable viscosity modifiers maybe used to adjust the viscosity of abio-ink. It should be noted, however, that certain ink formulations maynot require addition of any such viscosity modifiers, so long as theviscosity of the ink composition is already at or near a recommendedrange.

Typically, aqueous bio-ink compositions of the present invention containbetween about 0.1-35 vol % of viscosity modifying agent or agents in theformulation. In a broad sense, a viscosity modifying agent suitable foruse in water-based inks is a water-soluble solvent that regulates orcontributes to viscosity control in the liquid ink. In some embodiments,the provided bio-ink compositions contain between about 0.5-30%, about1.0-25%, about 5-20% of viscosity modifying agent agents (measured byvolume). In some embodiments, the provided bio-ink compositions containabout about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 18%, about 20%, about 21%, about 22%, about 23%, about24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%,about 31%, about 32%, about 33%, about 34%, about 35%, of viscositymodifying agent agents (measured by volume).

Aqueous bio-ink compositions disclosed described herein may include aviscosity modifier to modulate the final viscosity of the inkformulation. As already stated, any viscosity modifier known and used inthe pertinent art can be included in the bio-ink formulations providedherein.

In some embodiments, humectants may function as viscosity modifiers forthe bio-ink composition of the invention. Generally, a humectant is awater soluble solvent and any one of a group of hygroscopic substanceswith hydrating properties, i.e., used to keep things moist. They oftenare a molecule with several hydrophilic groups, most often hydroxylgroups; however, amines and carboxyl groups, sometimes esterified, canbe encountered as well (its affinity to form hydrogen bonds withmolecules of water, is the crucial trait).

Non-limiting examples of some humectants include: propylene glycol(E1520), hexylene glycol, and butylene glycol; glyceryl triacetate(E1518); vinyl alcohol; neoagarobiose; Sugar alcohols/sugar polyols:glycerol/glycerin, sorbitol (E420), xylitol, maltitol (E965); polymericpolyols (e.g., polydextrose (E1200)); quillaia (E999); urea; aloe veragel; MP Diol; alpha hydroxy acids (e.g., lactic acid); and, honey. Thechemical compound lithium chloride is an excellent (but toxic)humectant, as well. Typically, humectants such as glycerol and ethyleneglycol are used in water-based inks to prevent the nozzle from clogging.

Examples of other viscosity modifiers that may be included in the inksinclude, but are not limited to: acrylate esters, acrylic esters,acrylic monomer, aliphatic mono acrylate, aliphatic mono methacrylate,alkoxylated lauryl acrylate, alkoxylated phenol acrylate, alkoxylatedtetrahydrofurfuryl acrylate, C₁₂-C₁₄ alkyl methacrylate, aromaticacrylate monomer, aromatic methacrylate monomer, caprolactone acrylate,cyclic trimethylol-propane formal acrylate, cycloaliphatic acrylatemonomer, dicyclopentadienyl methacrylate, diethylene glycol methyl ethermethacrylate, epoxidized soybean fatty acid esters, epoxidized linseedfatty acid esters, epoxy acrylate, epoxy (meth)acrylate,2-(2-ethoxy-ethoxy) ethyl acrylate, ethoxylated (4) nonyl phenolacrylate, ethoxylated (4) nonyl phenol methacrylate, ethoxylated nonylphenol acrylate, glucose, fructose, corn syrup, gum syrup,hydroxy-terminated epoxidized 1,3-polybutadiene, isobornyl acrylate,isobornyl methacrylate, isodecyl acrylate, isodecyl methacrylate,isooctyl acrylate, isooctyl methacrylate, lauryl acrylate, laurylmethacrylate, methoxy polyethylene glycol (350) monoacrylate, methoxypolyethylene glycol (350) monomethacrylate, methoxy polyethylene glycol(550) monoacrylate, methoxy polyethylene glycol (550) monomethacrylate,nonyl-phenyl polyoxyethylene acrylate, octyldecyl acrylate,2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, polybutadienepolymer, polyester acrylate, polyester methacrylate, polyether acrylate,polyether methacrylate, polysorbates, stearyl acrylate, stearylmethacrylate, syrups, tetrahydrofurfuryl acrylate, tetrahydrofurfurylmethacrylate, triethylene glycol ethyl ether methacrylate,3,3,5-trimethylcyclohexyl methacrylate, urethane acrylate and urethanemethacrylate and combinations thereof.

Surfactants

In some embodiments, provided aqueous bio-ink compositions may contain asurfactant agent which works as a wetting and/or penetrating agent. Useof surfactants in water-based inks is, in some embodiments, crucialbecause even a relatively small amount of a surfactant can significantlymodify or affect the surface tension of an aqueous solution (e.g., wateror buffers). In some embodiments, a surfactant agent is present atconcentrations ranging between about 0.05-20%, e.g., between about0.1-10% (either by volume or by weight) of an ink composition.

Relative Ratios of Ink Components

These components are present in predetermined ratios.

To illustrate: in some embodiments, where a 6.25% silk fibroin solutionis used as the starting component (i), silk fibroin solution, anamphiphilic agent (such as a polysorbate) and water are present in avolume ratio of about 17:2:1. In some embodiments, silk fibroinsolution, an amphiphilic agent and water are present in a volume ratioof about 17:1.5:1.5. In some embodiments, silk fibroin solution, anamphiphilic agent and water are present in a volume ratio of about18:1.5:0.5. In some embodiments, silk fibroin solution, an amphiphilicagent and water are present in a volume ratio of about 16:2:2. In someembodiments, silk fibroin solution, an amphiphilic agent and water arepresent in a volume ratio of about 16:1.5:2.5. In some embodiments, silkfibroin solution, an amphiphilic agent and water are present in a volumeratio of about 16:2.5:1.5. In some embodiments, silk fibroin solution,an amphiphilic agent and water are present in a volume ratio of about16:3:1. In some embodiments, silk fibroin solution, an amphiphilic agentand water are present in a volume ratio of about 15:3:2. In someembodiments, silk fibroin solution, an amphiphilic agent and water arepresent in a volume ratio of about 15:2.5:2.5. In some embodiments, silkfibroin solution, an amphiphilic agent and water are present in a volumeratio of about 15:2:3. Of course, these ratios should be appropriatelyadjusted when as silk fibroin solution used as the starting component(i) contains different silk fibroin concentrations, such as 5%, 6%, 7%,8%, 9% and 10%.

In some embodiments, silk fibroin solutions used to prepare silk inksare substantially free of sericin (i.e., degummed). In some embodiments,silk fibroin useful for the preparation of silk inks described hereinare extracted from cocoons (i.e., natural source of silk fibers). Insome embodiments—silk fibroin useful for the preparation of silk inksdescribed herein are recombinantly produced. In some embodiments—silkfibroin useful for the preparation of silk inks described herein are lowmolecular weight silk fibroin.

Amphiphilic agents useful for preparing silk fibroin-based inks includesurfactants. In some embodiment, non-ionic detergents are used as anamphiphilic agent for this purpose. In some embodiments, silkfibroin-based inks (i.e., silk inks) comprise at least one polysorbate.Non-limiting examples of polysorbates include but are not limited to:polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or anycombinations thereof.

Additives, Dopants, and Biologically Active Agents

In any of the embodiments described herein, bio-ink compositions of thepresent invention may further include one or more agent(s) (e.g.,dopants and additives) suitable for intended purposes, includingtherapeutics (e.g., biologically active agents) and biological samples.Typically, addition of such agents (or dopants) are said to“functionalize” the ink composition by providing added functionality. Toprepare functional bio-inks by adding appropriate dopants, suitabledopants (one dopant or a combination of compatible dopants) may be mixedin with an ink (or to a component of such ink). Non-limiting examples ofsuitable agents (or dopants) to be added for functionalization ofbio-inks include but are not limited to: conductive or metallicparticles; inorganic particles; dyes/pigments; drugs (e.g., antibiotics,small molecules or low molecular weight organic compounds); proteins andfragments or complexes thereof (e.g., enzymes, antigens, antibodies andantigen-binding fragments thereof); cells and fractions thereof (virusesand viral particles; prokaryotic cells such as bacteria; eukaryoticcells such as mammalian cells and plant cells; fungi).

In some embodiments, the additive is a biologically active agent. Theterm “biologically active agent” as used herein refers to any moleculewhich exerts at least one biological effect in vivo. For example, thebiologically active agent can be a therapeutic agent to treat or preventa disease state or condition in a subject. Biologically active agentsinclude, without limitation, organic molecules, inorganic materials,proteins, peptides, nucleic acids (e.g., genes, gene fragments, generegulatory sequences, and antisense molecules), nucleoproteins,polysaccharides, glycoproteins, and lipoproteins. Classes ofbiologically active compounds that can be incorporated into thecomposition described herein include, without limitation, anticanceragents, antibiotics, analgesics, anti-inflammatory agents,immunosuppressants, enzyme inhibitors, antihistamines, anti-convulsants,hormones, muscle relaxants, antispasmodics, ophthalmic agents,prostaglandins, anti-depressants, anti-psychotic substances, trophicfactors, osteoinductive proteins, growth factors, and vaccines.

In some embodiments, the additive is a therapeutic agent. As usedherein, the term “therapeutic agent” means a molecule, group ofmolecules, complex or substance administered to an organism fordiagnostic, therapeutic, preventative medical, or veterinary purposes.As used herein, the term “therapeutic agent” includes a “drug” or a“vaccine.” This term include externally and internally administeredtopical, localized and systemic human and animal pharmaceuticals,treatments, remedies, nutraceuticals, cosmeceuticals, biologicals,devices, diagnostics and contraceptives, including preparations usefulin clinical and veterinary screening, prevention, prophylaxis, healing,wellness, detection, imaging, diagnosis, therapy, surgery, monitoring,cosmetics, prosthetics, forensics and the like. This term can also beused in reference to agriceutical, workplace, military, industrial andenvironmental therapeutics or remedies comprising selected molecules orselected nucleic acid sequences capable of recognizing cellularreceptors, membrane receptors, hormone receptors, therapeutic receptors,microbes, viruses or selected targets comprising or capable ofcontacting plants, animals and/or humans. This term can alsospecifically include nucleic acids and compounds comprising nucleicacids that produce a therapeutic effect, for example deoxyribonucleicacid (DNA), ribonucleic acid (RNA), nucleic acid analogues (e.g., lockednucleic acid (LNA), peptide nucleic acid (PNA), xeno nucleic acid(XNA)), or mixtures or combinations thereof, including, for example, DNAnanoplexes, siRNA, microRNA, shRNA, aptamers, ribozymes, decoy nucleicacids, antisense nucleic acids, RNA activators, and the like. Generally,any therapeutic agent can be included in the composition describedherein.

The term “therapeutic agent” also includes an agent that is capable ofproviding a local or systemic biological, physiological, or therapeuticeffect in the biological system to which it is applied. For example, thetherapeutic agent can act to control infection or inflammation, enhancecell growth and tissue regeneration, control tumor growth, act as ananalgesic, promote anti-cell attachment, and enhance bone growth, amongother functions. Other suitable therapeutic agents can includeanti-viral agents, hormones, antibodies, or therapeutic proteins. Othertherapeutic agents include prodrugs, which are agents that are notbiologically active when administered but, upon administration to asubject are converted to biologically active agents through metabolismor some other mechanism. Additionally, a silk-based drug deliverycomposition can contain one therapeutic agent or combinations of two ormore therapeutic agents.

A therapeutic agent can include a wide variety of different compounds,including chemical compounds and mixtures of chemical compounds, e.g.,small organic or inorganic molecules; saccharines; oligosaccharides;polysaccharides; biological macromolecules, e.g., peptides, proteins,and peptide analogs and derivatives; peptidomimetics; antibodies andantigen binding fragments thereof; nucleic acids; nucleic acid analogsand derivatives; an extract made from biological materials such asbacteria, plants, fungi, or animal cells; animal tissues; naturallyoccurring or synthetic compositions; and any combinations thereof. Insome embodiments, the therapeutic agent is a small molecule.

As used herein, the term “small molecule” can refer to compounds thatare “natural product-like,” however, the term “small molecule” is notlimited to “natural product-like” compounds. Rather, a small molecule istypically characterized in that it contains several carbon-carbon bonds,and has a molecular weight of less than 5000 Daltons (5 kDa), preferablyless than 3 kDa, still more preferably less than 2 kDa, and mostpreferably less than 1 kDa. In some cases it is preferred that a smallmolecule have a molecular weight equal to or less than 700 Daltons.

Exemplary therapeutic agents include, but are not limited to, thosefound in Harrison's Principles of Internal Medicine, 13th Edition, Eds.T. R. Harrison et al. McGraw-Hill N.Y., NY; Physicians' Desk Reference,50th Edition, 1997, Oradell N.J., Medical Economics Co.; PharmacologicalBasis of Therapeutics, 8th Edition, Goodman and Gilman, 1990; UnitedStates Pharmacopeia, The National Formulary, USP XII NF XVII, 1990, thecomplete contents of all of which are incorporated herein by reference.

Therapeutic agents include the herein disclosed categories and specificexamples. It is not intended that the category be limited by thespecific examples. Those of ordinary skill in the art will recognizealso numerous other compounds that fall within the categories and thatare useful according to the present disclosure. Examples include aradiosensitizer, a steroid, a xanthine, a beta-2-agonist bronchodilator,an anti-inflammatory agent, an analgesic agent, a calcium antagonist, anangiotensin-converting enzyme inhibitors, a beta-blocker, a centrallyactive alpha-agonist, an alpha-1-antagonist, ananticholinergic/antispasmodic agent, a vasopressin analogue, anantiarrhythmic agent, an antiparkinsonian agent, anantiangina/antihypertensive agent, an anticoagulant agent, anantiplatelet agent, a sedative, an ansiolytic agent, a peptidic agent, abiopolymeric agent, an antineoplastic agent, a laxative, anantidiarrheal agent, an antimicrobial agent, an antifungal agent, avaccine, a protein, or a nucleic acid. In a further aspect, thepharmaceutically active agent can be coumarin, albumin, steroids such asbetamethasone, dexamethasone, methylprednisolone, prednisolone,prednisone, triamcinolone, budesonide, hydrocortisone, andpharmaceutically acceptable hydrocortisone derivatives; xanthines suchas theophylline and doxophylline; beta-2-agonist bronchodilators such assalbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol;antiinflammatory agents, including antiasthmatic anti-inflammatoryagents, antiarthritis antiinflammatory agents, and non-steroidalantiinflammatory agents, examples of which include but are not limitedto sulfides, mesalamine, budesonide, salazopyrin, diclofenac,pharmaceutically acceptable diclofenac salts, nimesulide, naproxene,acetaminophen, ibuprofen, ketoprofen and piroxicam; analgesic agentssuch as salicylates; calcium channel blockers such as nifedipine,amlodipine, and nicardipine; angiotensin-converting enzyme inhibitorssuch as captopril, benazepril hydrochloride, fosinopril sodium,trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride,and moexipril hydrochloride; beta-blockers (i.e., beta adrenergicblocking agents) such as sotalol hydrochloride, timolol maleate, esmololhydrochloride, carteolol, propanolol hydrochloride, betaxololhydrochloride, penbutolol sulfate, metoprolol tartrate, metoprololsuccinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprololfumarate; centrally active alpha-2-agonists such as clonidine;alpha-1-antagonists such as doxazosin and prazosin;anticholinergic/antispasmodic agents such as dicyclomine hydrochloride,scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate,and oxybutynin; vasopressin analogues such as vasopressin anddesmopressin; antiarrhythmic agents such as quinidine, lidocaine,tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamilhydrochloride, propafenone hydrochloride, flecainide acetate,procainamide hydrochloride, moricizine hydrochloride, and disopyramidephosphate; antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa,selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, andbromocryptine; antiangina agents and antihypertensive agents such asisosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol andverapamil; anticoagulant and antiplatelet agents such as Coumadin,warfarin, acetylsalicylic acid, and ticlopidine; sedatives such asbenzodiazapines and barbiturates; ansiolytic agents such as lorazepam,bromazepam, and diazepam; peptidic and biopolymeric agents such ascalcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin,insulin, somatostatin, protirelin, interferon, desmopressin,somatotropin, thymopentin, pidotimod, erythropoietin, interleukins,melatonin, granulocyte/macrophage-CSF, and heparin; antineoplasticagents such as etoposide, etoposide phosphate, cyclophosphamide,methotrexate, 5-fluorouracil, vincristine, doxorubicin, cisplatin,hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase,altretamine, mitotane, and procarbazine hydrochloride; laxatives such assenna concentrate, casanthranol, bisacodyl, and sodium picosulphate;antidiarrheal agents such as difenoxine hydrochloride, loperamidehydrochloride, furazolidone, diphenoxylate hdyrochloride, andmicroorganisms; vaccines such as bacterial and viral vaccines;antimicrobial agents such as penicillins, cephalosporins, andmacrolides, antifungal agents such as imidazolic and triazolicderivatives; and nucleic acids such as DNA sequences encoding forbiological proteins, and antisense oligonucleotides.

Anti-cancer agents include alkylating agents, platinum agents,antimetabolites, topoisomerase inhibitors, antitumor antibiotics,antimitotic agents, aromatase inhibitors, thymidylate synthaseinhibitors, DNA antagonists, farnesyltransferase inhibitors, pumpinhibitors, histone acetyltransferase inhibitors, metalloproteinaseinhibitors, ribonucleoside reductase inhibitors, TNF alphaagonists/antagonists, endothelinA receptor antagonists, retinoic acidreceptor agonists, immuno-modulators, hormonal and antihormonal agents,photodynamic agents, and tyrosine kinase inhibitors.

Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin,netilmicin, streptomycin, amikacin, neomycin), bacitracin, corbapenems(e.g., imipenem/cislastatin), cephalosporins, colistin, methenamine,monobactams (e.g., aztreonam), penicillins (e.g., penicillin G,penicillinV, methicillin, natcillin, oxacillin, cloxacillin,dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin,piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, andvancomycin; and bacteriostatic agents such as chloramphenicol,clindanyan, macrolides (e.g., erythromycin, azithromycin,clarithromycin), lincomyan, nitrofurantoin, sulfonamides, tetracyclines(e.g., tetracycline, doxycycline, minocycline, demeclocyline), andtrimethoprim. Also included are metronidazole, fluoroquinolones, andritampin.

Enzyme inhibitors are substances which inhibit an enzymatic reaction.Examples of enzyme inhibitors include edrophonium chloride,N-methylphysostigmine, neostigmine bromide, physostigmine sulfate,tacrine, tacrine, 1-hydroxy maleate, iodotubercidin,p-bromotetramiisole, 10-(alpha-diethylaminopropionyl)-phenothiazinehydrochloride, calmidazolium chloride,hemicholinium-3,3,5-dinitrocatechol, diacylglycerol kinase inhibitor I,diacylglycerol kinase inhibitor II, 3-phenylpropargylamine,N°-monomethyl-Larginine acetate, carbidopa, 3-hydroxybenzylhydrazine,hydralazine, clorgyline, deprenyl, hydroxylamine, iproniazid phosphate,6-MeO-tetrahydro-9H-pyrido-indole, nialamide, pargyline, quinacrine,semicarbazide, tranylcypromine,N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride,3-isobutyl-1-methylxanthne, papaverine, indomethacind,2-cyclooctyl-2-hydroxyethylamine hydrochloride,2,3-dichloro-a-methylbenzylamine (DCMB),8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride, p-aminoglutethimide, p-aminoglutethimide tartrate, 3-iodotyrosine,alpha-methyltyrosine, acetazolamide, dichlorphenamide,6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.

Antihistamines include pyrilamine, chlorpheniramine, andtetrahydrazoline, among others.

Anti-inflammatory agents include corticosteroids, nonsteroidalanti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin,sulindac, tolmetin, ibuprofen, piroxicam, and fenamates), acetaminophen,phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexatecolchicine, allopurinol, probenecid, and sulfinpyrazone.

Muscle relaxants include mephenesin, methocarbomal, cyclobenzaprinehydrochloride, trihexylphenidyl hydrochloride, levodopa/carbidopa, andbiperiden.

Anti-spasmodics include atropine, scopolamine, oxyphenonium, andpapaverine.

Analgesics include aspirin, phenybutazone, idomethacin, sulindac,tolmetic, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin,morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids(e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate,loperamide, morphine sulfate, noscapine, norcodeine, normorphine,thebaine, nor-binaltorphimine, buprenorphine, chlomaltrexamine,funaltrexamione, nalbuphine, nalorphine, naloxone, naloxonazine,naltrexone, and naltrindole), procaine, lidocain, tetracaine anddibucaine.

Ophthalmic agents include sodium fluorescein, rose bengal, methacholine,adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase,betaxalol, pilocarpine, timolol, timolol salts, and combinations thereof

Prostaglandins are art recognized and are a class of naturally occurringchemically related long-chain hydroxy fatty acids that have a variety ofbiological effects.

Anti-depressants are substances capable of preventing or relievingdepression. Examples of anti-depressants include imipramine,amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine,doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazide.

Trophic factors are factors whose continued presence improves theviability or longevity of a cell. trophic factors include, withoutlimitation, platelet-derived growth factor (PDGP), neutrophil-activatingprotein, monocyte chemoattractant protein, macrophage-inflammatoryprotein, platelet factor, platelet basic protein, and melanoma growthstimulating activity; epidermal growth factor, transforming growthfactor (alpha), fibroblast growth factor, platelet-derived endothelialcell growth factor, insulin-like growth factor, glial derived growthneurotrophic factor, ciliary neurotrophic factor, nerve growth factor,bone growth/cartilage-inducing factor (alpha and beta), bonemorphogenetic proteins, interleukins (e.g., interleukin inhibitors orinterleukin receptors, including interleukin 1 through interleukin 10),interferons (e.g., interferon alpha, beta and gamma), hematopoieticfactors, including erythropoietin, granulocyte colony stimulatingfactor, macrophage colony stimulating factor and granulocyte-macrophagecolony stimulating factor; tumor necrosis factors, and transforminggrowth factors (beta), including beta-1, beta-2, beta-3, inhibin, andactivin.

Hormones include estrogens (e.g., estradiol, estrone, estriol,diethylstibestrol, quinestrol, chlorotrianisene, ethinyl estradiol,mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins(e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone,norgestrel), antiprogestin (mifepristone), androgens (e.g, testosteronecypionate, fluoxymesterone, danazol, testolactone), anti-androgens(e.g., cyproterone acetate, flutamide), thyroid hormones (e.g.,triiodothyronne, thyroxine, propylthiouracil, methimazole, andiodixode), and pituitary hormones (e.g., corticotropin, sumutotropin,oxytocin, and vasopressin). Hormones are commonly employed in hormonereplacement therapy and/or for purposes of birth control. Steroidhormones, such as prednisone, are also used as immunosuppressants andanti-inflammatories.

In some embodiments, the additive is an agent that stimulates tissueformation, and/or healing and regrowth of natural tissues, and anycombinations thereof. Agents that increase formation of new tissuesand/or stimulates healing or regrowth of native tissue at the site ofinjection can include, but are not limited to, fibroblast growth factor(FGF), transforming growth factor-beta (TGF-beta, platelet-derivedgrowth factor (PDGF), epidermal growth factors (EGFs), connective tissueactivated peptides (CTAPs), osteogenic factors including bonemorphogenic proteins, heparin, angiotensin II (A-II) and fragmentsthereof, insulin-like growth factors, tumor necrosis factors,interleukins, colony stimulating factors, erythropoietin, nerve growthfactors, interferons, biologically active analogs, fragments, andderivatives of such growth factors, and any combinations thereof.

In some embodiments, the silk composition can further comprise at leastone additional material for soft tissue augmentation, e.g., dermalfiller materials, including, but not limited to, poly(methylmethacrylate) microspheres, hydroxylapatite, poly(L-lactic acid),collagen, elastin, and glycosaminoglycans, hyaluronic acid, commercialdermal filler products such as BOTOX® (from Allergan), DYSPORT®,COSMODERM®, EVOLENCE®, RADIESSE®, RESTYLANE®, JUVEDERM® (from Allergan),SCULPTRA®, PERLANE®, and CAPTIQUE®, and any combinations thereof.

In some embodiments, the additive is a wound healing agent. As usedherein, a “wound healing agent” is a compound or composition thatactively promotes wound healing process. Exemplary wound healing agentsinclude, but are not limited to dexpanthenol; growth factors; enzymes,hormones; povidon-iodide; fatty acids; anti-inflammatory agents;antibiotics; antimicrobials; antiseptics; cytokines; thrombin;angalgesics; opioids; aminoxyls; furoxans; nitrosothiols; nitrates andanthocyanins; nucleosides, such as adenosine; and nucleotides, such asadenosine diphosphate (ADP) and adenosine triphosphate (ATP);neutotransmitter/neuromodulators, such as acetylcholine and5-hydroxytryptamine (serotonin/5-HT); histamine and catecholamines, suchas adrenalin and noradrenalin; lipid molecules, such assphingosine-1-phosphate and lysophosphatidic acid; amino acids, such asarginine and lysine; peptides such as the bradykinins, substance P andcalcium gene-related peptide (CGRP); nitric oxide; and any combinationsthereof.

In certain embodiments, the active agents described herein areimmunogens. In one embodiment, the immunogen is a vaccine. Most vaccinesare sensitive to environmental conditions under which they are storedand/or transported. For example, freezing may increase reactogenicity(e.g., capability of causing an immunological reaction) and/or loss ofpotency for some vaccines (e.g., HepB, and DTaP/IPV/HIB), or causehairline cracks in the container, leading to contamination. Further,some vaccines (e.g., BCG, Varicella, and MMR) are sensitive to heat.Many vaccines (e.g., BCG, MMR, Varicella, Meningococcal C Conjugate, andmost DTaP-containing vaccines) are light sensitive. See, e.g., Galazkaet al., Thermostability of vaccines, in Global Programme for Vaccines &Immunization (World Health Organization, Geneva, 1998); Peetermans etal., Stability of freeze-dried rubella virus vaccine (Cendehill strain)at various temperatures, 1 J. Biological Standardization 179 (1973).Thus, the compositions and methods described herein also provide forstabilization of vaccines regardless of the cold chain and/or otherenvironmental conditions.

In some embodiments, the additive is a cell, e.g., a biological cell.Cells useful for incorporation into the composition can come from anysource, e.g., mammalian, insect, plant, etc. In some embodiments, thecell can be a human, rat or mouse cell. In general, cells to be usedwith the compositions described herein can be any types of cells. Ingeneral, the cells should be viable when encapsulated withincompositions. In some embodiments, cells that can be used with thecomposition include, but are not limited to, mammalian cells (e.g. humancells, primate cells, mammalian cells, rodent cells, etc.), avian cells,fish cells, insect cells, plant cells, fungal cells, bacterial cells,and hybrid cells. In some embodiments, exemplary cells that can be canbe used with the compositions include platelets, activated platelets,stem cells, totipotent cells, pluripotent cells, and/or embryonic stemcells. In some embodiments, exemplary cells that can be encapsulatedwithin compositions include, but are not limited to, primary cellsand/or cell lines from any tissue. For example, cardiomyocytes,myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes,embryonic stem cells, adult stem cells, hematopoietic stem cells,hematopoietic cells (e.g. monocytes, neutrophils, macrophages, etc.),ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts,neurons, sperm cells, egg cells, liver cells, epithelial cells fromlung, epithelial cells from gut, epithelial cells from intestine, liver,epithelial cells from skin, etc, and/or hybrids thereof, can be includedin the silk/platelet compositions disclosed herein. Those skilled in theart will recognize that the cells listed herein represent an exemplary,not comprehensive, list of cells. Cells can be obtained from donors(allogenic) or from recipients (autologous). Cells can be obtained, as anon-limiting example, by biopsy or other surgical means known to thoseskilled in the art.

In some embodiments, the cell can be a genetically modified cell. A cellcan be genetically modified to express and secrete a desired compound,e.g. a bioactive agent, a growth factor, differentiation factor,cytokines, and the like. Methods of genetically modifying cells forexpressing and secreting compounds of interest are known in the art andeasily adaptable by one of skill in the art.

Differentiated cells that have been reprogrammed into stem cells canalso be used. For example, human skin cells reprogrammed into embryonicstem cells by the transduction of Oct3/4, Sox2, c-Myc and Klf4 (JunyingYu, et. al., Science, 2007, 318, 1917-1920 and Takahashi K. et. al.,Cell, 2007, 131, 1-12).

Pigment/Dye

In some embodiments, bio-ink compositions provided herein can include acolorant, such as a pigment or dye or combination thereof. Any organicand/or inorganic pigments and dyes can be included in the inks.Exemplary pigments suitable for use in the present invention includeInternational Color Index or C.I. Pigment Black Numbers 1, 7, 11 and 31,C.I. Pigment Blue Numbers 15, 15:1, 15:2, 15:3, 15:4, 15:6, 16, 27, 29,61 and 62, C.I. Pigment Green Numbers 7, 17, 18 and 36, C.I. PigmentOrange Numbers 5, 13, 16, 34 and 36, C.I. Pigment Violet Numbers 3, 19,23 and 27, C.I. Pigment Red Numbers 3, 17, 22, 23, 48:1, 48:2, 57:1,81:1, 81:2, 81:3, 81:5, 101, 114, 122, 144, 146, 170, 176, 179, 181,185, 188, 202, 206, 207, 210 and 249, C.I. Pigment Yellow Numbers 1, 2,3, 12, 13, 14, 17, 42, 65, 73, 74, 75, 83, 93, 109, 110, 128, 138, 139,147, 142, 151, 154 and 180, D&C Red No. 7, D&C Red No. 6 and D&C Red No.34, carbon black pigment (such as Regal 330, Cabot Corporation),quinacridone pigments (Quinacridone Magenta (228-0122), available fromSun Chemical Corporation, Fort Lee, N.J.), diarylide yellow pigment(such as AAOT Yellow (274-1788) available from Sun ChemicalCorporation); and phthalocyanine blue pigment (such as Blue 15:3(294-1298) available from Sun Chemical Corporation). The classes of dyessuitable for use in present invention can be selected from acid dyes,natural dyes, direct dyes (either cationic or anionic), basic dyes, andreactive dyes. The acid dyes, also regarded as anionic dyes, are solublein water and mainly insoluble in organic solvents and are selected, fromyellow acid dyes, orange acid dyes, red acid dyes, violet acid dyes,blue acid dyes, green acid dyes, and black acid dyes. European Patent0745651, incorporated herein by reference, describes a number of aciddyes that are suitable for use in the present invention. Exemplaryyellow acid dyes include Acid Yellow 1 International Color Index or C.I.10316); Acid Yellow 7 (C.I. 56295); Acid Yellow 17 (C.I. 18965); AcidYellow 23 (C.I. 19140); Acid Yellow 29 (C.I. 18900); Acid Yellow 36(C.I. 13065); Acid Yellow 42 (C.I. 22910); Acid Yellow 73 (C.I. 45350);Acid Yellow 99 (C.I. 13908); Acid Yellow 194; and Food Yellow 3 (C.I.15985). Exemplary orange acid dyes include Acid Orange 1 (C.I. 13090/1);Acid Orange 10 (C.I. 16230); Acid Orange 20 (C.I. 14603); Acid Orange 76(C.I. 18870); Acid Orange 142; Food Orange 2 (C.I. 15980); and Orange B.

Exemplary red acid dyes include Acid Red 1. (C.I. 18050); Acid Red 4(C.I. 14710); Acid Red 18 (C.I. 16255), Acid Red 26 (C.I. 16150); AcidRed 2.7 (C.I. as Acid Red 51 (C.I. 45430, available from BASFCorporation, Mt. Olive, N.J.) Acid Red 52 (C.I. 45100); Acid Red 73(C.I. 27290); Acid Red 87 (C. I. 45380); Acid Red 94 (C.I. 45440) AcidRed 194; and Food Red 1 (C.I. 14700). Exemplary violet acid dyes includeAcid Violet 7 (C.I. 18055); and Acid Violet 49 (C.I. 42640). Exemplaryblue acid dyes include Acid Blue 1 (C.I. 42045); Acid Blue 9 (C.I.42090); Acid Blue 22 (C.I. 42755); Acid Blue 74 (C.I. 73015); Acid Blue93 (C.I. 42780); and Acid Blue 158A (C.I. 15050). Exemplary green aciddyes include Acid Green 1 (C.I. 10028); Acid Green 3 (C.I. 42085); AcidGreen 5 (C.I. 42095); Acid Green 26 (C.I. 44025); and Food Green 3 (C.I.42053). Exemplary black acid dyes include Acid Black 1 (C.I. 20470);Acid Black 194 (Basantol® X80, available from BASF Corporation, anazo/1:2 CR-complex.

Exemplary direct dyes for use in the present invention include DirectBlue 86 (C.I. 74180); Direct Blue 199; Direct Black 168; Direct Red 253;and Direct Yellow 107/132 (C.I. Not Assigned).

Exemplary natural dyes for use in the present invention include Alkanet(C.I. 75520, 75530); Annafto (C.I. 75120); Carotene (C.I. 75130);Chestnut; Cochineal (C.I. 75470); Cutch (C.I. 75250, 75260); Divi-Divi;Fustic (C.I. 75240); Hypernic (C.I. 75280); Logwood (C.I. 75200); OsageOrange (C.I. 75660); Paprika; Quercitron (C.I. 75720); Sanrou (C.I.75100); Sandal Wood (C.I. 75510, 75540, 75550, 75560); Sumac; andTumeric (C.I. 75300). Exemplary reactive dyes for use in the presentinvention include Reactive Yellow 37 (monoazo dye); Reactive Black 31(disazo dye); Reactive Blue 77 (phthalo cyanine dye) and Reactive Red180 and Reactive Red 108 dyes. Suitable also are the colorants describedin The Printing Ink Manual (5th ed., Leach et al. eds. (2007), pages289-299. Other organic and inorganic pigments and dyes and combinationsthereof can be used to achieve the colors desired.

In addition to or in place of visible colorants, bio-ink compositionsdescribed herein can contain UV fluorophores that are excited in the UVrange and emit light at a higher wavelength (typically 400 nm andabove). Examples of UV fluorophores include but are not limited tomaterials from the coumarin, benzoxazole, rhodamine, napthalimide,perylene, benzanthrones, benzoxanthones or benzothia-xanthones families.The addition of a UV fluorophore (such as an optical brightener forinstance) can help maintain maximum visible light transmission. Theamount of colorant, when present, generally is between 0.05% to 5% orbetween 0.1% and 1% based on the weight of the bio-ink composition.

For non-white inks, the amount of pigment/dye generally is present in anamount of from at or about 0.1 wt % to at or about 20 wt % based on theweight of the ink composition. In some applications, a non-white ink caninclude 15 wt % or less pigment/dye, or 10 wt % or less pigment/dye or 5wt % pigment/dye, or 1 wt % pigment/dye based on the weight of the inkcomposition. In some applications, a non-white ink can include 1 wt % to10 wt %, or 5 wt % to 15 wt %, or 10 wt % to 20 wt % pigment/dye basedon the weight of the ink composition. In some applications, a non-whiteink can contain an amount of dye/pigment that is 1 wt %, 2 wt %, 3 wt %,4 wt %, 5%, 6 wt %, 7 wt %, 8 wt %, 9 wt %, 10 wt %, 11 wt %, 12 wt %,13 wt %, 14 wt %, 15%, 16 wt %, 17 wt %, 18 wt %, 19 wt % or 20 wt %based on the weight of the ink composition.

For white ink compositions, the amount of white pigment generally ispresent in an amount of from at or about 1 wt % to at or about 60 wt %based on the weight of the ink composition. In some applications,greater than 60 wt % white pigment can be present. Preferred whitepigments include titanium dioxide (anatase and rutile), zinc oxide,lithopone (calcined coprecipitate of barium sulfate and zinc sulfide),zinc sulfide, blanc fixe and alumina hydrate and combinations thereof,although any of these can be combined with calcium carbonate. In someapplications, a white ink can include 60 wt % or less white pigment, or55 wt % or less white pigment, or 50 wt % white pigment, or 45 wt %white pigment, or 40 wt % white pigment, or 35 wt % white pigment, or 30wt % white pigment, or 25 wt % white pigment, or 20 wt % white pigment,or 15 wt % white pigment, or 10 wt % white pigment, based on the weightof the ink composition. In some applications, a white ink can include 5wt % to 60 wt %, or 5 wt % to 55 wt %, or 10 wt % to 50 wt %, or 10 wt %to 25 wt %, or 25 wt % to 50 wt %, or 5 wt % to 15 wt %, or 40 wt % to60 wt % white pigment based on the weight of the ink composition. Insome applications, a non-white ink can an amount of dye/pigment that is5%, 6 wt %, 7 wt %, 8 wt %, 9 wt %, 10 wt %, 11 wt %, 12 wt %, 13 wt %,14 wt %, 15%, 16 wt %, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22wt %, 23 wt %, 24 wt %, 25%, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt%, 31 wt %, 32 wt %, 33 wt %, 34 wt %, 35%, 36 wt %, 37 wt %, 38 wt %,39 wt %, 40 wt %, 41 wt %, 42 wt %, 43 wt %, 44 wt %, 45%, 46 wt %, 47wt %, 48 wt %, 49 wt %, 50 wt %, 51 wt %, 52 wt %, 53 wt %, 54 wt %,55%, 56 wt %, 57 wt %, 58 wt %, 59 wt % or 60 wt % based on the weightof the ink composition.

The lifetime (e.g., stability) of provided bio-ink compositions dependson the usage and the storage conditions. In some embodiments, storage ina refrigerator at 4 degree C. when finishing printing is recommended. Insome embodiments, provided bio-inks (with our without dopants) may bestored without refrigeration, such as at room temperature (typicallybetween about 18-26° C.) for an extended duration of time withoutsignificant loss of function. In some embodiments, provided bio-inks(with our without dopants) may be stored at room temperature (typicallybetween about 18-26° C.) for an extended duration of time, such as atleast for 1 week, at least for 2 weeks, at least for 3 weeks, at leastfor 4 weeks, at least for 6 weeks, at least for 2 months, at least for 3months, at least for 4 months, at least for 5 months, at least for 6months, at least for 9 months, at least for 12 months, at least for 15months, at least for 18 months, and at least for 24 months, or longer,without significant loss of function. In some embodiments, providedbio-inks (with our without dopants) may be stored at elevatedtemperature (between about 27-40° C.) for at least part of the durationof storage, for an extended duration of time, such as at least for 1week, at least for 2 weeks, at least for 3 weeks, at least for 4 weeks,at least for 6 weeks, at least for 2 months, at least for 3 months, atleast for 4 months, at least for 5 months, at least for 6 months, atleast for 9 months, at least for 12 months, at least for 15 months, atleast for 18 months, and at least for 24 months, or longer, withoutsignificant loss of function.

Properties of Bio-Inks

According to some example embodiments of the present invention, providedbio-ink compositions have suitable properties as measured by surfacetension, viscosity and/or pH. In some embodiments, a bio-ink compositionof the present disclosure is prepared as described herein, so that theink composition has, for example, surface tension ranges between about24-50 dynes/cm at room temperature, between about 28-44 dynes/cm at roomtemperature, between about 30-38 dynes/cm at room temperature; viscosityof between about 8-10 centipoise at room temperature. In someembodiments, such ink composition has a pH value of between about 5-9,such as between 6-7, between 5.5-7.5. As used herein, “room temperature”is typically between about 18-26° C., and also referred to as “ambient”condition. In some embodiments, therefore, the phrase “room temperature”and “ambient temperature (or condition)” may include temperatures suchas about 18° C., about 19° C., about 20° C., about 21° C., about 22° C.,about 23° C., about 24° C., about 25° C., about 26° C., and so on,unless otherwise specified.

Preparation of Bio-Inks

Another aspect of the invention provides methods for preparing bio-inks,such as silk fibroin inks. An exemplary protocol for preparing a silkfibroin ink in accordance with the present disclosure is provided below.

Preparation of Low Molecular Weight Structural Proteins

While silk fibroin extraction methods generally have been welldocumented, the present invention encompasses the recognition thatcertain structural proteins can be processed further to be made suitablefor bio-printing described herein, thereby overcoming previously existedhurdles that had prevented the use of certain structural proteins forprinting purposes.

Accordingly, in some embodiments, such methods involve extraction ofstructural proteins (such as silk fibroin) under high temperature, suchas between about 101-135° C., between about 105-130° C., between about110-130° C., between about 115-125° C., between about 118-123° C., e.g.,about 115° C., 116° C., 117° C., 118° C., 119° C., 120° C., 121° C.,122° C., 123° C., 124° C., 125° C.

Additionally or alternatively, provided methods in some embodimentsinvolve extraction of structural proteins (such as silk fibroin) underelevated pressure, such as about 5 psi, 6 psi, 7 psi, 8 psi, 9 psi, 10psi, 11 psi, 12 psi, 13 psi, 14 psi, 15 psi, 16 psi, 17 psi, 18 psi, 19psi, 20 psi, 21 psi, 22 psi, 23 psi, 24 psi, 25 psi, 30 psi, 31 psi, 32psi, 33 psi, 34 psi and 35 psi. In some embodiments, structural proteins(such as silk fibroin) are extracted under high temperature and underelevated pressure, e.g., at about 110-130° C. and about 10-20 psi for aduration suitable to produce a protein solution that would easily gothrough a 0.2 μm filter. In some embodiments, structural proteins (suchas silk fibroin) are extracted under high temperature and under elevatedpressure, e.g., at about 110-130° C. and about 10-20 psi for about60-180 minutes. In some embodiments, structural proteins (such as silkfibroin) are extracted under high temperature and under elevatedpressure, e.g., at about 116-126° C. and about 12-20 psi for about90-150 minutes.

An exemplary protocol is provided below:

In accordance with some examples, the following example process may beperformed to obtain ˜40 mL of silk solution with a concentration of˜6.25% (wt/vol); if more volumes are needed, the materials can be scaledappropriately.

1) Cut Bombyx mori silk cocoons (10 gram) into half-dime-sized piecesand dispose of silkworms;

2) Measure 8.48 gram of sodium carbonate and add it into 4 liter ofwater in a 5 liter glass beaker (to prepare a 0.02 M solution);

3) Put the beaker into an autoclave and set the autoclave to run at 121degree C. under the pressure of 16 psi for 120 minutes;

4) Remove the silk fibroin with a strainer and cool it by rinsing inultrapure cold water for 20 minutes and repeat twice for a total ofthree rinses;

5) After the third rinse, remove the silk fibroin and squeeze the water;

6) Spread the squeezed silk fibroin, spread it out and let it dry in afume hood for 12 hours, which results in silk fibroin weighing slightlyover 2.5 gram;

7) Dissolve 2.5 gram of silk fibroin into 10 mL of 9.3 M lithiumbromide;

8) The silk fibroin should dissolve completely in a few minutes uponstirring;

9) Insert 10 mL of the silk-LiBr solution into a pre-wet 3-12-mLdialysis cassette and dialyze against 1 liter of ultrapure water for 48hours (change the water every 6 hours);

10) Remove silk from the cassette;

11) Place the silk solution in a centrifuge and spin at 9,000 r.p.m. at2 degree C. for 60 minutes, and store the centrifuged silk solution (˜40mL of silk solution with a concentration of ˜6.25%) in a refrigerator at4 degree C.

For ˜2 mL of silk fibroin protein ink, if more volumes are needed, thematerials can be scaled appropriately.

1) Mix the silk solution with surfactant (for example, Tween 20 fromSigma-Aldrich Co.) and water in a volume ratio of 17:2:1 (i.e. 1700 μLof ˜6.25% silk fibroin solution, 200 μL of Tween 20 and 100 μL ofwater);

2) This results in a 2 mL of silk fibroin ink with the followingproperties:

-   -   Surface tension: 30-38 dynes/cm at room temperature    -   Viscosity: 8-10 centipoise at room temperature    -   pH value: between 6 and 7.        Note that the ratio of the mixture is optimized for Tween 20 and        other biological or chemical surfactant (for example, glycol,        ether, and etc.) can be also used with modifications of the        mixture ratio. Surface treatment of the printing nozzle(s) can        also improve the formation of silk ink drops.        Silk as Biomaterial

Even among structural proteins, silk fibroin (SF) is a fascinatingmaterial, extensively investigated for its potential in textile,biomedical, photonic and electronic applications. SF is a structuralprotein, like collagen, but with a unique feature: it is produced fromthe extrusion of an amino-acidic solution by a living complex organism(while collagen is produced in the extracellular space by self-assemblyof cell-produced monomers). SF properties are derived from itsstructure, which consists of hydrophobic blocks staggered byhydrophilic, acidic spacers. In its natural state, SF is organized inβ-sheet crystals alternated with amorphous regions, which providestrength and resilience to the protein. The multiplicities of forms inwhich regenerated SF can be process at a high protein concentration andmolecular weight make it attractive for several high-tech applications,as recently discovered and described by our group. The degree ofcrystallinity of the protein can be finely tuned and it influences SF'sbiological, physical, biochemical and mechanical properties. Inaddition, the amino-acidic nature of SF brings a diversity of side chainchemistries that allows for the incorporation and stabilization ofmacromolecules useful in drug delivery applications or in providingcellular instructions. In particular, we have recently showed that drySF with diverse degrees of crystallinity stabilizes vaccines andantibiotics, eliminating the need for the cold chain. SF is indeedconsidered a platform technology in biomaterials fabrication as itsrobustness and qualities bring the assets to add a large portfolio ofdistinct features (e.g. nanopatterning, biochemical functionalization)to the final construct. Processing of regenerated SF generally involvesthe partial or total dehydration of a fibroin solution (protein contentof 1-15 wt %) to form films, sponges, gels, spheres (micron- tonano-sized) and foams with numerous techniques (e.g. solvent casting,freeze drying, salt leaching, sonication). The rationale beyond thesefabrication processes is to manufacture a robust material that combinesmechanical strength with biochemical properties.

Liquid Droplet Unit

The present invention provides means for achieving a very small unitvolume of a bio-ink composition formed as liquid droplets for carryingout printing. In some embodiments, the present application provides anaqueous unit composition (i.e., a liquid droplet) having a volume ofbetween about 0.1-100 pL. In some embodiments, each droplet has a volumeof between about 0.5-50 pL, between about 0.5-25 pL, between about0.5-20 pL, between about 0.5-15 pL, between about 0.5-10 pL, betweenabout 1.0-40 pL, between about 1.0-30 pL, between about 1.0-25 pL,between about 1.0-20 pL, between about 1.0-15 pL, between about 1.0-10pL. For example, in some embodiments, a unit volume of a single dropletof a bio-ink described herein is about 0.5 pL, about 1.0 pL, about 1.5pL, about 2.0 pL, about 3.0 pL, about 4.0 pL, about 5.0 pL, about 6.0pL, about 7.0 pL, about 8.0 pL, about 9.0 pL, about 10 pL, about 11 pL,about 12 pL, about 13 pL, about 14 pL, about 15 pL, about 16 pL, about17 pL, about 18 pL, about 19 pL, about 20 pL, about 21 pL, about 22 pL,about 23 pL, about 24 pL, about 25 pL, about 30 pL, about 40 pL, about50 pL, about 60 pL, about 70 pL, about 80 pL, about 90 pL, or about 100pL. In some embodiments, for example, each droplet of such a bio-ink hasa volume of between 0.1-100 pL, wherein the bio-ink has a viscosityranging between 8-10 centipoise at room temperature.

Uniformity

The ability to control droplet size distribution (e.g., uniformity) onthe micron scale and sub-micron (e.g., nano) scale to ensurereproducible printing quality is important in a number of contexts. Forexample, for downstream applications involving medical or clinical use,such as drug delivery applications, it is crucial to ensure theuniformity of release kinetics. Generally, monodisperse particles (e.g.,droplets) provide greater degree of uniformity in release kinetics thanpolydisperse particles (e.g., droplets). Similarly, it is of particularinterest to have the ability to finely control droplet size anddistributions in a number of optical applications involving the use ofnano-sized particles, such as the plasmonic resonance of core/shellnanospheres and their tunable, size-dependent, optical properties.Moreover, the use of protein-based materials provides additional utilityfor these systems, since they can be absorbed by the body with safedegradation.

The process of “break-off” and jetting of the droplet from a nozzle maybe at least in part determined by, or otherwise influenced by interplayof a number of factors, including the interfacial tension and theviscosity of the liquid ink, among other factors. This, in conjunctionwith the nozzle characteristic of the printer, in turn determines thesize of the droplets that breaks off and to be deposited onto asubstrate. Moreover, interactions between a bio-ink and a substrate(e.g., charge interactions) also affects the behavior.

Accordingly, as a result of extensive experimentation, the inventors ofthe present application have determined certain desirable sets ofconditions and/or parameters by which to produce reproducibly uniformdroplet formation and high quality printing, as exemplified below.

As used herein, the term “uniform” or “uniformity” refers to acomposition characterized by a plurality of units of similar featureswith respect to a parameter (such as size, e.g., volume and diameter).The less the degree of deviation with respect to a parameter beingmeasured, the greater the degree of uniformity within the composition.

In some embodiments, for example, dots of a bio-ink described herein areuniform in that liquid ink droplets deposited for printing show a narrowsize distribution such that a majority of droplets within a singleprinting run fall within a specified range of volumes.

In some embodiments, at least 50% of droplets have volumes within aspecified range, wherein the specified range may be between about 1.0 pLand 20 pL. In some embodiments, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% or greater number of droplets within a single printingrun have volumes within a specified range. The specified range may bebetween about 10 pL and about 20 pL.

In some embodiments, provided aqueous bio-inks have a useful viscosityrange of about 5-15 centipoise at jetting temperature. In someembodiments, provided aqueous bio-inks have a useful viscosity range ofabout 8-14 centipoise, e.g., about 8-13 centipoise, about 7-13centipoise, about 6-12 centipoise, about 6-11 centipoise, about 7-12centipoise, about 9-13 centipoise, about 10-13 centipoise, about 10-12centipoise, for example, about 5 centipoise, about 6 centipoise, about 7centipoise, about 8 centipoise, about 9 centipoise, about 10 centipoise,about 11 centipoise, about 12 centipoise, about 13 centipoise, about 14centipoise, about 15 centipoise at jetting temperature.

It has been further determined that, in any one of the embodiments,provided aqueous bio-inks have a useful surface tension range of about20-50 dynes/cm at jetting temperature. In some embodiments, providedaqueous bio-inks have a useful surface tension range of about 22-48dynes/cm, about 23-47 dynes/cm, about 24-46 dynes/cm, about 25-47dynes/cm, about 26-46 dynes/cm, about 27-45 dynes/cm, 28-44 dynes/cm,for example, about 28 dynes/cm, about 29 dynes/cm, about 30 dynes/cm,about 31 dynes/cm, about 32 dynes/cm, about 33 dynes/cm, about 34dynes/cm, about 35 dynes/cm, about 36 dynes/cm, about 37 dynes/cm, about38 dynes/cm, about 39 dynes/cm, about 40 dynes/cm, about 41 dynes/cm,about 42 dynes/cm, about 43 dynes/cm and about 44 dynes/cm at jettingtemperature.

In addition, extensive work has revealed that the parameter of viscosityand surface tension are co-dependent, such that relatively highersurface tension may be well combined with relatively lower viscosity toachieve effective printing. For example, a bio-ink having a surfacetension greater than 44 can combine well with a viscosity lower than 10in the context of DMP 2800 printer by way of modifying the waveform.

It has been further determined that, in any one of the embodiments,provided aqueous bio-inks exhibit low volatility, such that suchbio-inks preferably have boiling point higher than 100° C., e.g., about100° C., 101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107° C.,108° C., 109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115° C.,116° C., 117° C., 118° C., 119° C., 120° C., 121° C., 122° C., 123° C.,124° C., 125° C., 126° C., 127° C., 128° C., 129° C., 130° C., orgreater.

Further yet, it has been determined that, in any one of the embodiments,provided aqueous bio-inks have specific gravity greater than 1.0.

It has also been determined that, in any of the embodiments, providedaqueous bio-ink compositions have a useful pH range of between about 4and 9, e.g., about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5,about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0,about pH 8.5, about pH 9.0. In some embodiments, a buffer component maybe added to such ink compositions to maintain the pH level in the ink.For example, in some embodiments, buffering salt components mayoptionally comprise between about 0.1-0.5% (by weight) of bio-inkcompositions.

Low Molecular Weight Structural Proteins

According to the invention, structural proteins particularly suitablefor formulating as a bio-ink are below 200 kDa, preferably below 150kDa. In some embodiments, structural proteins (or fragments thereof)particularly suitable for formulating as a bio-ink have molecular weightranging between about 3.5 kDa and about 120 kDa, e.g., about 3.5-110kDa, about 3.5-100 kDa, about 3.5-90 kDa, about 3.5-80 kDa, about 3.5-70kDa, about 3.5-60 kDa, about 3.5-50 kDa, about 3.5-40 kDa, about 3.5-35kDa, about 3.5-30 kDa, about 3.5-25 kDa, about 3.5-20 kDa, about 50-120kDa, about 60-120 kDa, about 70-120 kDa, about 80-120 kDa, about 90-120kDa. In some embodiments, such structural protein may be a full-length(e.g., wild type) structural protein having a molecular weight fallingwithin any of the ranges shown above. In other embodiments, suchstructural protein may be so-called “low molecular weight protein,”i.e., corresponding to reduced size fragments of a full-lengthcounterpart, for example fragments of the full-length counterpart.

In some embodiments, where silk fibroin is used as a structural proteinfor a bio-ink, no more than 15% of the total number of silk fibroinfragments in a silk fibroin ink composition has a molecular weightexceeding 200 kDa, and at least 50% of the total number of the silkfibroin fragments in the population has a molecular weight within aspecified range, wherein the specified range is between about 3.5 kDaand about 120 kDa. Low molecular weight silk fibroin is described indetail in U.S. provisional application 61/883,732, entitled “LOWMOLECULAR WEIGHT SILK FIBROIN AND USES THEREOF,” the entire contents ofwhich are incorporated herein by reference.

In some embodiments, consistency of a bio-ink may be further enhanced byselectively enriching certain range or ranges of fragment size(molecular weight) in a preparation. In some embodiments, therefore, astep of filtration may be included during the preparation of such an inkcomposition. For instance, filters with a known cut-off range (such as0.2 μm) may be used to remove any fragments or aggregates (e.g.,contamination) that are larger than the pore size. Alternatively oradditionally, in some embodiments, protein solution may be furtherprocessed, including extended heating and/or high pressure treatment, inorder to promote fragmentation of large structural proteins.

In some embodiments, an aqueous protein ink solution described hereinmay be heated (such as by boiling at atmospheric pressure) during theprocess of protein preparation for a period of time, e.g., for about 10minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40minutes, about 45 minutes, about 50 minutes, about 60 minutes, about 70minutes, about 80 minutes, about 90 minutes, about 100 minutes, about110 minutes, about 120 minutes, or longer.

Alternatively or additionally, in some embodiments, such proteinsolution may be heated or boiled at an elevated temperature. Forexample, in some embodiments, the protein solution can be heated orboiled at about 101.0° C., at about 101.5° C., at about 102.0° C., atabout 102.5° C., at about 103.0° C., at about 103.5° C., at about 104.0°C., at about 104.5° C., at about 105.0° C., at about 105.5° C., at about106.0° C., at about 106.5° C., at about 107.0° C., at about 107.5° C.,at about 108.0° C., at about 108.5° C., at about 109.0° C., at about109.5° C., at about 110.0° C., at about 110.5° C., at about 111.0° C.,at about 111.5° C., at about 112.0° C., at about 112.5° C., at about113.0° C., 113.5° C., at about 114.0° C., at about 114.5° C., at about115.0° C., at about 115.5° C., at about 116.0° C., at about 116.5° C.,at about 117.0° C., at about 117.5° C., at about 118.0° C., at about118.5° C., at about 119.0° C., at about 119.5° C., at about 120.0° C.,or higher.

In some embodiments, such elevated temperature can be achieved bycarrying out at least portion of the heating process (e.g., boilingprocess) under pressure. For example, suitable pressure under whichprotein fragments described herein can be produced are typically betweenabout 10-40 psi, e.g., about 11 psi, about 12 psi, about 13 psi, about14 psi, about 15 psi, about 16 psi, about 17 psi, about 18 psi, about 19psi, about 20 psi, about 21 psi, about 22 psi, about 23 psi, about 24psi, about 25 psi, about 26 psi, about 27 psi, about 28 psi, about 29psi, about 30 psi, about 31 psi, about 32 psi, about 33 psi, about 34psi, about 35 psi, about 36 psi, about 37 psi, about 38 psi, about 39psi, or about 40 psi.

Alternatively or additionally, in some embodiments, protein solution maybe further processed, including centrifugation.

In some embodiments, provided aqueous bio-inks have low dissolved gascontents. In some embodiments, a step of degassing may be optionallyperformed prior to printing in order to enhance printing quality.

It should be noted that certain structural proteins, including silkfibroin, exhibit an inherent self-assembly property. In someembodiments, this process involves the formation of beta-sheet secondarystructure within a structural protein (or fragments). As such, bio-inkscomprising a structural protein described herein may contain a range ofdegrees/levels of beta-sheet crystallinity. For example, providedprotein ink compositions may contain a beta-sheet content rangingbetween about 5% and 70%, e.g., about 5%, about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65% or about 75%.

Printed Arrays

One aspect of the invention relates to printed forms of biopolymer-basedinks (bio-inks). Typically, such prints are made by the use of at leastone bio-ink described herein and include an array of unit dots formedfrom ink droplets, deposited upon a substrate. Thus, in someembodiments, a printed array comprises a substrate; and a plurality ofdot units, wherein the plurality of dot units has a semi-solid or solidform, wherein each dot unit comprises a low molecular weight structuralprotein. Typically, each dot unit on the substrate is between about0.1-250 μm in diameter. According to the invention, a plurality of dotunits is deposited upon a substrate in a predetermined spatial patternto form a structure, e.g., 2D structures and 3D structures.

Thus, inkjet printers such as those described herein may be used toprint biopolymer patterns, such as, for example, dots, signal line, and2D patterns, on both hydrophilic and hydrophobic substrates. Theresolution of printing is affected by viscosity and surface tension ofthe biopolymer ink. Also, the resolution of pattern may depend on theroughness of the substrate and nozzle size of the printer. An exampleprinter that provides a 10 pl size nozzle to make patterns produces adrop size around 25 μm and the width of a printed line which is around40 um on the hydrophilic substrates. A signal layer line will give theinterface between dots, and a 2D pattern presents interface betweenlines.

Parameter Setting for Biopolymer Ink

Voltage

The voltage is a function of drop size and drop velocity. So the voltagesetting depends on the desired height level of the nozzle above thesubstrate and the desired drop size to be printed. However, withinsufficient voltage (in some examples, voltage level below 15 V), thebiopolymer ink will not come out due to the surface tension ofbiopolymer ink. Higher voltage settings, on the other hand, increase thevolume of the drop. FIG. 21 shows biopolymer lines on a silicon waferunder 15 v, 20V and 25 v voltage printing, with the width of thebiopolymer lines being 65 μm, 100 μm, and 110 μm. As shown, high voltageprinting gives greater-width lines due to increasing the drop volume.

Cleaning Cycle

Before printing, a purging process may be performed, which applies airpressure to the outside of a fluid bag to force fluid through the entirefluid path and out all nozzles, as shown in the waveform of FIG. 22.After the purging process, air in the chamber is forced out of thenozzles, and ensures ink wets the nozzle to start printing.

During printing, a blotting process may be provided to absorb biopolymerink in close proximity to the nozzle plate. After the blotting process,the excess biopolymer ink, which may cause misdirected firing, isremoved.

After printing, a spitting process may protect the nozzle from clogging.The spitting process provides for ejection of some drops ink from thechamber. This allows the fresh biopolymer drops to reach the meniscus toreplace the previous one from the prior drop.

Nozzle Number

The number of nozzle may also affect the printing patterns. FIG. 23shows a biopolymer line on acrylic with 25 v and one nozzle printing,and provides a 40 μm width biopolymer line. FIG. 24 shows a biopolymerline with a 240 μm—although still under 25 v printing, the width of theline is substantially increased in comparison to the pattern of FIG. 23,because of printing by seven nozzles as opposed to one.

Biopolymer Drops from Nozzle

FIG. 25 shows the drops from 10 pL nozzles, and the voltage value set at23V, with jetting frequency at 5 KHz. The uniform drops from the nozzleshow stable performance. There are no misdirected nozzles which meansthat biopolymer solution jetting smooth without bubbles under the highfrequency oscillating system. All of the sixteen nozzles in this examplework well for at least 8 hours which means the high temperaturebiopolymer will not clog the 20 μm diameter nozzle.

Various Biopolymer Patterns Using Direct Inkjet Printing Technique

Dots

FIG. 26 and FIG. 27 show biopolymer dots printed on silicon wafer andacrylic, respectively. This example utilizes 1 nozzle and 1-layerprinting, the voltage value is 15 v and the jetting frequency is 1 KHz.The size of dots is 40 μm on silicon wafer (FIG. 26) and 30 μm onacrylic (FIG. 27).

Lines

Multiple Layers Printing

FIG. 28 shows biopolymer lines which are printed with one nozzle and 15v on the silicon wafer. FIG. 29 shows the SEM image of this one-layerprinting. One-layer printing is clear without any interface betweendrops. Comparing one-layer printing with three-layer printing, one-layerpatterns are more uniform and the edge of line is cleaner. A rough edgeis present in this example on three-layers printing (FIG. 30), becausethe upper layer fluid causes capillary instability when the upper layersof biopolymer are printed. FIG. 31 shows the first line as being widerthan the other four lines, because the alignment of first line is not asgood as the other four lines. FIG. 32 shows serious capillaryinstability in a twenty-layer pattern, so multiple-layer printing may bebest suited, in some examples, for low resolution patterns.

Cross Lines Printing

In accordance with example embodiments, the method for printing multiplelayer lines and cross lines is different. For multiple layer lines, thesubstrate is fixed during printing and the direction of printing amongmultiple layer lines is the same. For the cross lines printing, thesubstrate is rotated by 90 degree C. after first layer printing, andthen the second layer printing is performed. As such, the direction ofthe two layers is different. FIGS. 33 to 35 indicate capillaryinstability between two layers, and the edge of pattern shows a cleangradual capillary instability process.

Two Dimension Printing

A one-layer square pattern shows an interface between lines. As shown inthe example of FIG. 36, there is less than 1 μm width overlap betweentwo lines. After applying a laser point to the pattern, a diffractiongrating pattern shows on the wall due to the 1 μm overlap, as shown inFIG. 37. However, the overlap part of the pattern disappears afterprinting the second layer pattern. As such, the multiple layers give asmooth finish pattern (FIGS. 38 and 39).

Biopolymer Patterns after Alcohol Annealing Treatments

Biopolymer film is easily dissolved in water. However it will notdissolve after alcohol annealing due to the formation of β sheet. Aprinter may be used to make a β sheet pattern. To do so, the printingpattern is set to do a 2-hour vacuum annealing. It turns out that, insome examples, the patterns tend to spread out, as shown in FIG. 40,which shows the biopolymer pattern before annealing and after annealing.

Thickness of Biopolymer Pattern

Biopolymers, such as, for example, silk, provide a biologicallyfavorable environment allowing them to entrain various biological andchemical dopants and maintain their functionality. Mixing differentchemical solutions with biopolymer solution gives different viscosityand surface tension which affect the thickness of pattern. As is clear,the number of printing layers is another important element that affectsthe thickness of pattern. In an example implementation, three kinds ofbiopolymer solution are prepared and include food color biopolymer, highrefractive index biopolymer, and pure biopolymer (e.g., pure silk), andthen printed with a number of nozzles. FIGS. 41 to 43 show the thicknessof patterns are increased by the number printing layer. The thinnestpattern in this example is less than 100 nm created by a one-layer foodcolor biopolymer pattern. According to FIG. 44, the thickest pattern ispure biopolymer pattern due to highest percentage biopolymer in thesolution.

Biopolymer Patterns on Various Substrates

The printable substrates for biopolymer ink may include, for example,paper, glass, silicon, metals, cloth textiles, and plastics. Suchsubstrates can be divided into two groups which are hydrophobicsubstrates and hydrophilic substrates. The drop size on hydrophobicsubstrate is smaller due to high surface energy. The width of biopolymerlines from FIG. 28 is similar with respect to the biopolymer lines fromFIG. 45. However, the two patterns are supplied by different voltages.Biopolymer patterns on silicon have slightly larger voltage values.

Direct Printing of Functional Biopolymer Devices Using Doped BiopolymerSolution as the Ink

A biopolymer such as, for example, Silk fibroin is shown to be effectivematerial and matrix that can maintain the functionalities of dopants.Therefore, choosing the appropriate dopants (including both physicaldopants, e.g. metallic nanoparticles, laser dyes, quantum dots, etc.,and biochemical dopants, e.g., cells, enzymes, bacterium, etc.) andmixing them into silk fibroin solution or other biopolymer solution asthe ink is an advantageous way to directly print functional devicesusing an inkjet printer. In the following section, a series offunctional biopolymer devices (with different dopants) are described asexamples.

Inkjet Printing of Gold Nanoparticle Doped Biopolymer Patterns

As mentioned above, biopolymers provide a biologically favorableenvironment allowing them to entrain various biological and chemicaldopants and maintain their functionality. Proteins and enzymes have beendoped into various biopolymer material formats, especially biopolymerfilms. Biopolymer films have been doped with gold nanoparticles suchthat they resonantly absorb incident light and convert the light toheat, which may used as, for example, a biocompatible thermal therapyfor in vivo medical applications such as killing of tumor tissue andbacteria.

The preparation of gold nanoparticle biopolymer ink includes theproduction of the print grade biopolymer film (e.g., silk fibroinsolution) and synthesis of gold nanoparticles, followed by a simplemixing of the two in solution with a certain ratio that is determined byapplication. Where silk fibroin solution is utilized, pre-cut Bombyxmori cocoon pieces are boiled in a 0.02 M Na₂CO₃ solution for 2 hours toremove sericin, and boiled silk fibers are dried overnight and thendissolved in a 9.3 M LiBr at 60 degree C. for 4 hours. The lithiumbromide salt is then removed from the silk solution through awater-based dialysis process. The gold nanoparticle solution is preparedby adding 20 mL 1% Na₃C₆H₅O₇ into 200 mL boiled 1.0 mM HAuCl₄, followedby continuously heating for 10 minutes until the solution has turneddeep red. Then the gold nanoparticle solution is carefully added intothe silk solution with gentle agitation for uniform dispersion and isready for printing after being filtered, e.g., against a 0.2 micronfilter.

Table 1 provides the main parameters for printing and the printingresult is shown in FIG. 47.

The printed Au-NPs doped biopolymer device shows enhanced plasmaticabsorption of green light (FIG. 48), resulting in a temperature increaseof ˜15 degree s with an irradiance of ˜0.25 W/cm². The heating effectscould be further improved and optimized by adjusting the Au-NPsconcentration and layers of the printed structures, which could bepotentially used for light-mediated patterned heating treatments.

TABLE 1 Printing Permanents for Gold Nanoparticle Biopolymer Ink Voltage25 v Nozzle Number 4 Drop Spacing 25 μm Printing Layer 5 FiringFrequency  2 KHzInkjet Printing of Enzyme Doped Biopolymer Patterns

In addition to printing gold nanoparticle doped biopolymer, it is alsopossible to directly print enzyme-doped biopolymer for biomedicalapplications such as enzyme-linked immunosorbant assay test (i.e.ELISA). ELISA is a widely used test to identify certain substances usingantibodies and the colorimetric change as the sensing/diagnosticmechanism. Usually, the enzymes used in ELISA tests need to be stored atlow temperature for maintaining the bioactivities. It has been shownthat biopolymer can help to maintain the functionalities of the dopedenzyme at room temperatures without fridge-storage. Therefore, directlyprinting of enzyme-doped biopolymer patterns (in a precise way) is anadvantageous mechanism for applications such as, for example, rapid andlow volume screening tests, food allergens, and toxicology applications,as shown in FIG. 49.

Inkjet Printing of Antibiotics Doped Biopolymer Patterns

The use of antibiotics is important for effective infectious diseasecontainment and curing. However, most, if not all, current antibioticsneed to be maintained within a specific refrigeration temperature rangedue to their temperature sensitivity. Silk fibroin, as an example, hasbeen proven to be a biologically friendly protein polymer. Recently,researchers found that silk was capable of stabilizing labileantibiotics (in the form of films) even at temperatures up to 60 degreeC. over more than 6 months. Direct inkjet printing of antibiotics-dopedbiopolymer by mixing penicillin solution of various concentration levelswith purified biopolymer solution prepared as previously described maybe provided. Compared to antibiotics-doped biopolymer films, directprinting of antibiotics-doped biopolymer has the advantages of precisecontrol of the antibiotic distribution and potential multilayer andmulti-drugs printing that may benefit, for example, more sophisticatedcases where fine control and micro-manipulation of the antibiotic drugare needed.

To obtain a clear pattern on bacterial growth area, first we use methodone, below, to print a pattern before bacterial growth. The result showsthat two clean squares without any clean pattern in the Petri dish after5 hours incubation, as shown in FIG. 50.

Method One:

1) Culture 50 ul bacterial on agar

2) Print 1 layer an arrow and text pattern on bacterial, with drop gap50 um

3) Take 5 hours culture in 37 degree C. incubator

To improve the method, the pattern is printed after bacterial overnightgrowth, as provided by method two, below. There is an arrow in the inthe Petri dish after 9 hours incubation (FIG. 51).

Method Two:

1) Culture bacterial on agar

2) Take overnight culture in 37 degree C. incubator

3) Print 2 layers an arrow and 25 um drop gap on bacterial

4) Take 9 hours culture in 37 degree C. incubator

Inkjet Printing of Colored Biopolymer Patterns

In accordance with example embodiments, in addition to biomedicalapplications (e.g., implantable biomedical applications), biopolymersare used to construct edible food sensors as a green and edible materialthat is extracted and purified from domesticated silkworm cocoons. Plainbiopolymer solution (i.e. non-doped biopolymer solution) is a water-likehighly transparent protein solution that is colorless.

Food coloring, alternatively called color additive, imparts color whenadded to food or drink, and is used widely both in commercial foodproduction and in domestic cooking. Commercially available food dyes(considered as safe) may be mixed with biopolymer solution to makecolored biopolymer inks, e.g., for direct inkjet printing. Further,patterns may be printed on textile silk which carries a basic color(light yellow).

To achieve a clear pattern on textile silk, multiple-layer printing maybe beneficial, because the color of textile silk is darker than a blankpaper. After seven layers of printing, the pattern is clear andbeautiful, as shown in FIG. 52.

The colored silk patterns remain in their original patterns after 2hours of vacuum annealing. The patterns also survive a dry cleaningprocess, as shown in FIG. 53.

Multiple-color biopolymer printing may benefit from an alignmentprocess, because the printer in accordance with some examples, loads onecartridge with one color at a time, as shown in FIG. 54. In someexamples, there are four steps of alignment including multiple-layeralignment, cartridge, voltage alignment, and nozzle alignment.

Multilayer alignment: one color for each layer of printing;

Cartridge alignment: set drop offset before every layer printing;

Voltage alignment: different color inks have slight change in viscosity;

Nozzle alignment: using the same nozzles for every layer printing(number of nozzles determines line width).

Direct printing of silk fibroin protein based inks using a inkjetprinters (e.g., commercially available inkjet printers) has been shownand described. Various types of biopolymer inks may be prepared bychoosing appropriate dopants and mixing with the biopolymer solution(e.g., purified silk fibroin solution), and printed. A set of operatingparameters may be optimized for each individual biopolymer ink(including, for example, gold nanoparticle biopolymer ink, enzyme-dopedbiopolymer ink, high refractive index biopolymer ink, and antibacterialbiopolymer ink) to improve the performance for specific applications.Both single layer and multiple-layer printing may be carried out, withadvantageous resolutions (e.g., a resolution of 25 microns).

Resolution

One aspect of the invention relates to printed forms of biopolymer-basedinks (bio-inks). Typically, such prints made by the use of at least onebio-inks described herein include an array of unit dots formed from inkdroplets, deposited upon a substrate. Thus, in some embodiments, aprinted array comprises a substrate; and a plurality of dot units,wherein the plurality of dot units has a semi-solid or solid form,wherein each dot unit comprises a low molecular weight structuralprotein. Typically, each dot unit on the substrate is between about0.1-250 μm in diameter. According to the invention, a plurality of dotunits is deposited upon a substrate in a predetermined spatial patternto form a structure, e.g., 2D structures and 3D structures. Typically,printed forms of biopolymer-based inks prepared in accordance with thedisclosure of the present application have a resolution of between about50-20,000 dpi, e.g., about 100 dpi, about 200 dpi, about 300 dpi, about400 dpi, about 500 dpi, about 600 dpi, about 700 dpi, about 800 dpi,about 900 dpi, about 1000 dpi, about 1100 dpi, about 1200 dpi, about1500 dpi, about 2000 dpi, about 2500 dpi, about 3000 dpi, about 3500dpi, about 4000 dpi, about 4500 dpi, about 5000 dpi, about 5500 dpi,about 6000 dpi, about 6500 dpi, about 7000 dpi, about 7500 dpi, about8000 dpi, about 8500 dpi, about 9000 dpi, about 9500 dpi, about 10000dpi, about 11000 dpi, about 12000 dpi, about 13000 dpi, about 14000 dpi,about 15000 dpi, about 16000 dpi, about 17000 dpi, about 18000 dpi,about 19000 dpi, and about 20000 dpi.

Substrates

A variety of substrates may be suitable for use in printing a bio-inkdescribed herein. Such printable substrates using bio-inks arelimitless, simply depending on the available inkjet printers.Non-limiting examples of useful substrates include, but are not limitedto: papers, polyimide, polyethylene, natural fabric, synthetic fabric,metals, liquid crystal polymer, palladium, glass and other insulators,silicon and other semiconductors, metals, cloth textiles and fabrics,plastics, biological substrates, such as cells and tissues, protein- orbiopolymer-based substrates (e.g., agarose, collagen, gelatin, etc.),and any combinations thereof.

In some embodiments of the invention, provided bio-inks can be printedon substrates that generally are of a flexible material, such as aflexible polymer film or paper, such as wax paper or non-wax substrates.In some embodiments, suitable substrates include releasable substrates,such as a label release grade or other polymer coated paper, as is knownin the art (e.g., see 6,939,576). Such substrate also can be or includea non-silicone release layer. Such substrate also can be a plastic orpolymer film, such as any one of an acrylic-based film, apolyamide-based film, a polyester-based film, a polyolefm-based filmsuch as polyethylene and polypropylene, a polyethylene naphthylene-basedfilm, a polyethylene terephthalate-based film, a polyurethane-based filmor a PVC-based film, or a combination thereof.

Printable Patterns and Structures

In some embodiments, inkjet printing can be employed for printingpatterns of bio-inks, such as silk fibroin inks (with or withoutdopants). The printable patterns (e.g., structures) using bio-inks arelimitless, simply depending on the available inkjet printers. Theprintable patterns (e.g., structures) include, but are not limited toregular and irregular patterns, such as lines, curves, dots, solids, andany combinations thereof.

Each pattern or structure to be printed is formed from a plurality ofsmall “dots” each of which is generated from a liquid droplet of abio-ink deposited onto the substrate.

Such patterns can be either one layer of dot prints or multilayer ofprints (e.g., serial printing), depending on the intended applications.Each layer in the multilayer prints can be overlapping on top of eachother for thicker patterns or cross with other layers for complicatedpatterns. In some embodiments, serial printing can be performed tofabricate a 3D structure.

Annealing and/or Crosslinking

As already mentioned, certain structural proteins, including silkfibroin, exhibit an inherent self-assembly property. In someembodiments, this process involves the formation of beta-sheet secondarystructure within a structural protein (or fragments). As such, bio-inkscomprising a structural protein described herein may contain a range ofdegrees/levels of beta-sheet crystallinity. For example, providedprotein ink compositions may contain a beta-sheet content rangingbetween about 5% and 70%, e.g., about 5%, about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65% or about 75%.

Depending on the applications in some embodiment, a conformationalchange can be induced in such structural protein or low molecular weightfragments thereof to control or tune the solubility of the protein-basedstructure printed on a substrate. In some embodiments, theconformational change can induce the protein at least partiallyinsoluble. Without wishing to be bound by a theory, the inducedconformational change alters the crystallinity of the protein, e.g.,beta-sheet crystallinity. In some embodiments, including with silkfibroin-based inkjet prints, the conformational change may be induced byany methods known in the art, including, but not limited to, alcoholimmersion (e.g., ethanol, methanol), water annealing, shear stress,ultrasound (e.g., by sonication), pH reduction (e.g., pH titrationand/or exposure to an electric field) and any combinations thereof. Forexample, the conformational change can be induced by one or moremethods, including but not limited to, controlled slow drying (Lu etal., Biomacromolecules 2009, 10, 1032); water annealing (Jin et al., 15Adv. Funct. Mats. 2005, 15, 1241; Hu et al; Biomacromolecules 2011, 12,1686); stretching (Demura & Asakura, Biotech & Bioengin. 1989, 33, 598);compressing; solvent immersion, including methanol (Hofmann et al., JControl Release. 2006, 111, 219), ethanol (Miyairi et al., J. Fermen.Tech. 1978, 56, 303), glutaraldehyde (Acharya et al., Biotechnol J.2008, 3, 226), and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)(Bayraktar et al., Eur J Pharm Biopharm. 2005, 60, 373); pH adjustment,e.g., pH titration and/or exposure to an electric field (see, e.g., U.S.Patent App. No. US2011/0171239); heat treatment; shear stress (see,e.g., International App. No.: WO 2011/005381), ultrasound, e.g.,sonication (see, e.g., U.S. Patent Application Publication No. U.S.2010/0178304 and International App. No. WO2008/150861); and anycombinations thereof. Contents of all of the references listed above areincorporated herein by reference in their entireties.

In some embodiments, the conformation of certain structural proteins ina bio-ink, including silk fibroin, may be altered by water annealing.Without wishing to be bound by a theory, it is believed that physicaltemperature-controlled water vapor annealing (TCWVA) provides a simpleand effective method to obtain refined control of the molecularstructure of biomaterials. To illustrate a non-limiting example, in thecase of silk fibroin, the relative degree of crystallinity can becontrolled, ranging from a low beta-sheet content using conditions at 4°C. (α helix dominated silk I structure), to higher beta-sheet content of60% crystallinity at 100° C. (β-sheet dominated silk II structure).Water or water vapor annealing is described, for example, in PCTapplication no. PCT/US2004/011199, filed Apr. 12, 2004 and no.PCT/US2005/020844, filed Jun. 13, 2005; and Jin et al., Adv. Funct.Mats. 2005, 15:1241 and Hu et al., Biomacromolecules, 2011, 12(5):1686-1696, contents of all of which are incorporated herein by referencein their entireties.

Alternatively or additionally, in some embodiments, alteration in theconformation of certain structural proteins, such as silk fibroin, maybe induced by immersing in alcohol or organic solvent, e.g., methanol,ethanol, propanol, acetone, etc. The alcohol concentration can be atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% or 100%. In someembodiment, alcohol concentration is 100%. If the alteration in theconformation is by immersing in a solvent, the protein composition canbe washed, e.g., with solvent/water gradient to remove any of theresidual solvent that is used for the immersion. The washing step can berepeated one, e.g., one, two, three, four, five, or more times.

Alternatively or additionally, the alteration in the conformation ofcertain structural proteins, such as silk fibroin, can be induced withshear stress. The shear stress can be applied, for example, by passing astructural protein composition through a needle. Other methods ofinducing conformational changes include applying an electric field,applying pressure, and/or changing the salt concentration.

The treatment time for inducing the conformational change can be anyperiod of time to provide a desired degree of beta-sheet crystallinitycontent. In some embodiments, the treatment time can range from about 1hour to about 12 hours, from about 1 hour to about 6 hours, from about 1hour to about 5 hours, from about 1 hour to about 4 hours, or from about1 hour to about 3 hours. In some embodiments, the sintering time canrange from about 2 hours to about 4 hours or from 2.5 hours to about 3.5hours.

When inducing the conformational change is by solvent immersion,treatment time can range from minutes to hours. For example, immersionin the solvent can be for a period of at least about 15 minutes, atleast about 30 minutes, at least about 1 hour, at least about 2 hours,at least 3 hours, at least about 6 hours, at least about 18 hours, atleast about 12 hours, at least about 1 day, at least about 2 days, atleast about 3 days, at least about 4 days, at least about 5 days, atleast about 6 days, at least about 7 days, at least about 8 days, atleast about 9 days, at least about 10 days, at least about 11 days, atleast about 12 days, at least about 13 days, or at least about 14 days.In some embodiments, immersion in the solvent can be for a period ofabout 12 hours to about seven days, about 1 day to about 6 days, about 2to about 5 days, or about 3 to about 4 days.

After the treatment to induce the conformational change, structuralproteins, such as silk fibroin, may comprise a beta-sheet crystallinitycontent of at least about 5%, at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, or at least about 90%.

Device (e.g., Printers)

It is desired, in accordance with example embodiments, to turn abiopolymer solution into the “ink” for directly printing biopolymerdevices using a suitable printer. It is very challenging though sincethere are certain requirements for the biopolymer to function as ink.Inkjet printers have grown in popularity and performance—actually,inkjet printers are by far the most popular since their introduction inthe latter half of the 1980s. Compared to laser printers (which use dryink, also known as toner, static electricity, and heat to print), inkjetprinters use liquid inks and nozzles (usually multiple nozzles needed)to spray drops of ink directly onto the substrates.

A typical inkjet printer includes: a) print head—that contains a seriesof nozzles that are used to spray the ink drops; b) ink cartridge—thatcontains the ink; c) stepper motor—that moves the print head back andforth across the substrate.

It is noted most inkjet printers use piezoelectric nozzle techniques forprecision printing. Such techniques use piezo crystals that vibrate whenthey receive a very small electric charge. When the crystal vibratesinward and outward, it pulls and forces a tiny amount of ink and spraysit out of the nozzle.

Referring to FIGS. 6 and 7 an inkjet printer is provided. The inkjetprinter may be, for example, a commercial inkjet printer, e.g., aFUJIFILM Dimatix Materials Printer DMP-2800. The illustrated printeruses piezoelectric inkjet technology and MEMS fabrication processes (forcartridges, nozzles and etc.).

Referring to FIG. 2, the printer includes a base, a print cartridge, amaintenance station blotting pad, a platen, a drop watcher, and a lid.

As shown in FIG. 2, the printer works, in this example, with a maximumprintable area of A4 size substrate (8×11 inch) with a disposable (butreusable with certain modifications/tweaks) piezo inkjet cartridge. Themaximum height of printable substrate is up to 25 mm in the illustratedexample. The printer also has the ability to heat up the substrate up to60 degrees C. In addition, there is a fiducially camera availableallowing real time watching the formation of the drop as it is ejectedfrom the nozzle.

The printer may serve as a convenient laboratory tool that enables users(e.g., students, researchers, and engineers) to evaluate the use ofspecific inks (e.g., silk fibroin solution in accordance with exampleembodiments) for new and proof of principle technology development withextensive flexibilities to optimize process parameters for user orientedapplications.

Following are some example applications using the printer and listins ofvarious types of functional materials and printable inks.

Printer-compatible substrates may include, for example: paper, Kapton(i.e. polyimide), poyethylene (PET), fabrics (such as, for example,cotton, nylon, polyester), metals (such as, for example, aluminum foil,copper foil, stainless steel foil and etc.), liquid crystal polymer,palladium, and glass.

Printer-compatible fluids/inks may include, for example: conductivesilvers, conductive inorganics (e.g., non silver ink, such as ITO inks),conductive organics (such as OLED), single wall carbon nanotubes(SWCNTs), insulators, polyimides, photoresists, resins, and UV curableinks.

Among different types of conductive inks, a wide range of ink propertiesincluding viscosity, density, surface tension, and dispersion stabilitymay be observed. Therefore, it may be necessary to optimize the printerparameters such as the volume of the jetted ink, the gap distancebetween droplets, the printing frequency, temperatures of the jetted inkand the substrate, and/or the sintering/curing mechanism performed afterprinting. A popular application of using inkjet printer for conductiveprinting is rapid printing of RFID tags. However, this is a ratherchallenging endeavor since precise control of the desired conductivityand pattern designs (on non-perfect substrates, for example, onnon-glossy rough papers).

Unlike the traditional photolithograph and etching PCB fabricationprocess (which is a subtractive method by removing undesired metal fromthe substrate surface), conductive inkjet printing for RF applicationsjets the single conductive ink droplet from the nozzle to a predefinedposition (usually controlled by, for example computer and a precisemotor stepper). Accordingly, no harsh chemicals as the etching waste arecreated, which results in an economical and ecological fabricationsolution. Silver nano-particle inks are selected and commonly used forgood metal conductivity. As mentioned above, a sintering process eitherby applying heat or UV exposure (to remove excess solvent and to removematerial impurities) is usually needed, which also enhance the bondstrength between the ink and the as-printed substrate. It should benoted that an immediate sintering process may be essential, because thebiopolymer ink may begin to oxidize, which would reduce the efficiencyof conductivity of the metallic patterns.

As shown in FIG. 8, there are, in the illustrated structure, gapsbetween printed silver nanoparticles after printing, resulting in a poorconnection and therefore lack of sufficient conductivity. After aperiod, e.g., 15 minutes, of a heating/curing process, the particlesbegin to aggregate and gaps start to diminish, which forms a continuousmetal film and guarantees a good conductor, which determines theperformance of printed electric devices such as, for example, an RFIDtag as shown in FIG. 9. The simulated and measured intensity overfrequency of an example embodiment in accordance with FIG. 9 is shown inFIG. 10. As shown in the graph, this example has a working frequency of2 GHz, although example embodiments may be configured to have anysuitable frequency range.

One potential aspect of inkjet printing techniques is “bioprinting”,which requires micro-level, and in many cases, nano-level, liquidmanipulation. Typical applications may include, for example,micro-dosing, biochemical surface patterning and modification, tissueengineering and importantly the direct placement of living cells, DNAarrays, and proteomics. Questions have been raised on the influence ofmechanical forces and relatively intense electric field during theinkjet process on cells and some research reports show that althoughsome cell death may occur, surviving cells recover rapidly and seem tobehave normally, as shown in FIG. 11.

Working Principle of Materials Printer

In accordance with example embodiments, materials printers (e.g., theDMP-2800 inkjet printer from FUJIFILM Dimatix, Inc.) are used todirectly print regenerated silk fibroin protein. The printers may beable, in some implementations, to create patterns and load BMP patternsover any suitable area (e.g., an area of about 200×300 mm). The printersmay allow substrates having any suitable dimensions, (e.g., substratesup to 25 mm thick or much thicker or thinner substrates) and may have anadjustable Z height.

The materials printers may have any suitable number of jetting nozzles,e.g., piezo-based jetting nozzles. For example, a materials printer mayhave a single nozzle or a plurality of any desired number of nozzles. Insome embodiments, between 8 and 32 nozzles are provided. For example,some embodiments have 16 jetting nozzles. Where a plurality of suchnozzles are provided, the nozzles may be arranged with any suitablespacing, which may be regular or irregular. For example, the nozzles maybe spaced from each at a spacing that is between 30 μm and 500 μm,between 50 μm and 400 μm, between 150 μm and 350 μm, or between 200 μmand 300 μm, e.g, at 254 μm spacing. The nozzle or nozzles may beprovided in connection with a fillable cartridge.

In some embodiments, a method is provided in which the biopolymer base(e.g., silk fibroin protein) is regenerated as the ink to match specificfluid requirement of the printer. The printed biopolymer (e.g., silkprotein) may be biocompatible and can be further functionalized bymixing the biopolymer ink with appropriate dopants (including, forexample, organic and/or inorganic dopants) for specific applications.

Printer Operation

The printer may serve, for example, as a powerful laboratory tool, andmay have capabilities to allow users to optimize process parameters,such as, for example, nozzle voltage, substrate height, and wave form.The printer may, in some embodiments, provide multi-layer printing andallow an alignment process when using multiple cartridges and matchingthe origin point on the substrate. In some example embodiments, theprinter operates according to a method including creating a pattern,loading ink, and setting printing permanents.

As indicated above, a printer may be utilized in an example method thatincludes creating a pattern, loading ink, and setting printingpermanents. The printer, in some implementations, may include aprocessor running software and that accepts pattern files and/orprovides a pattern creation and editing software interface to allowusers to create and/or edit patterns. The pattern creation and editingsoftware allows the user to modify a pattern of drops for printing,which may be useful for some fine and small scale patterns, for example,patterns illustrated in FIG. 12. However, creating a complex structurepattern for printing may be very time consuming. For patterns such asillustrated in FIG. 13, a transformation to a pattern design file formatmay first be required, followed by importation into the pattern designsoftware.

In some example embodiments, to print fine patterns, a user may firstmake a high resolution original file before transforming the file intothe file format of the pattern design and editing software, because somesuch software only allows importation of monochrome Bitmap files whichcan be a low resolution file. After creating a Bitmap pattern, a usermay select an option titled Pattern Editor (Bitmap images) from the mainsoftware window and choose a drop space size which depends on the inkand substrate before importing the Bitmap file. It may be beneficial forthe user to double check the final size of the pattern to minimize therisk of error. If the final size is not in accordance with the user'sexpectation, the user may adjust the final size by changing the patternsize in the Bitmap file, then reloading the pattern. The pattern may berepeated by controlling a placement number.

For a user to create his or her own pattern, the user may select anoption in the software, e.g., in a main windows of the software, andselect an option indicated as Pattern Edition. This allows the user tocreate a pattern by entering dimensions in a Pattern Block DropPositions field. Alternatively, the user may draw a feature through acorresponding software window. Before creating the pattern, the user maywish to choose the size of drop spacing which is the center to centerdistance from one drop to the next. For a normal biopolymer drop (e.g.,a silk drop) the drop spacing may be, for example, about 25 μm on ahydrophilic surface, such as, for example, a silicon wafer.

To load the cartridge, the ink used to fill into the cartridge may needgo through a filter first. For example, for a 26 μm nozzle and a dropvolume of 10 pL, the ink may pass through a 0.22 μm filter first. Inthis example, the nozzle may easy be clogged by particle sizes largerthan 0.22 μm.

After loading a new cartridge onto the printer, the user may change theclean pad for new ink to avoid contamination by other chemicals. Afterloading the cartridge, the user may select the pattern the use wishes toprint from the software. The system may automatically calculate thecartridge mounting angle determined by drop spacing specified in thepattern. Table 2 shows the relation of saber angle, resolution, and dropspacing.

TABLE 2 Resolutions relationships Resolution Saber angle Drop spacing[dpi] [°] [μm] 5080.00 1.1 5 2540 2.3 10 1693.33 3.4 15 1270.00 4.5 201016.00 5.6 25 846.67 6.8 30 725.71 7.9 35 635.00 9.1 40 564.44 10.2 45508.00 11.4 50 461.82 12.5 55 423.33 13.7 60 390.77 14.8 65 362.86 16.070 338.67 17.2 75 317.50 18.4 80 298.82 19.6 85 282.22 20.8 90 267.3722.0 95 254.00 23.2 100 241.90 24.4 105 230.91 25.7 110 220.87 26.9 115211.67 28.2 120 203.20 29.5 125 195.38 30.8 130 188.15 32.1 135 181..4333.4 140 175.17 34.8 145 169.33 36.2 150 163.87 37.6 155 158.75 39.0 160153.94 40.5 165 149.41 42.0 170 145.41 43.5 175 141.11 45.1 180 137.3046.7 185 133.68 48.4 190 130.26 50.1 195 127.00 51.9 200 123.90 53.8 205120.95 55.8 210 118.14 57.8 215 115.45 60.0 220 112.89 62.4 225 110.4364.9 230 108.09 67.7 235 105.83 70.9 240 103.67 74.7 245 101.60 79.8 250100.00 90 254

In some example embodiments, to set printing parameters, the user mayselect a drop watch button and the system may move the cartridge to theright side of the platen, positioning the nozzles over the drop watchercamera system. The user may first select the range of nozzles the userwishes to jet the pattern. Second, the user may modify the nozzles touniform performance by adjusting voltage of those nozzle as beingmonitored by camera. Another potentially important parameter the usermay wish to set up carefully is the cartridge print height according tothe substrate thickness. If the substrate thickness is, for example,less than 0.5 mm, the printable range may be 210 mm*315 mm. If thesubstrate thickness is, for example, from 0.5 mm to 25 mm, the printablerange may be 210 mm*260 mm. The repeatability distance may be, forexample, ±25 μm. The last step before printing may be an alignmentprocess through a fiducial camera tool, e.g., via a tools menu in thesoftware window. First, the user may calibrate the position of a newcartridge or head angle by setting the drop offset to automatic ormanual, e.g., from the tools menu. Second, the user may set the printingorigin point and reference point for multiple-layer printing.

Setting Cartridge Parameters

In some implementations, to optimize drop performance, there are someparameters that may need be set up precisely. The main parameters mayinclude, for example, nozzle voltage, nozzle temperature, meniscus setpoint, cleaning cycles, and waveform.

To adjust nozzle voltage, the user may click on an edition button thecartridge setting box of the software. In some examples, the voltage ofeach nozzle may be individually adjusted, as shown in FIG. 14.Increasing voltage increases drop volume and jetting velocity. Asuitable velocity to be set is, for example, 7-9 m/sec.

To adjust nozzle temperature, meniscus set point, nozzle number, andprint height, the user may make a selection in the cartridge settings ofthe software.

The user may lower the ink viscosity and surface tension by increasingnozzle temperature. The printer may allow the user to adjust nozzletemperature in a range from, for example, 28 degree C. to 70 degree C.,as shown in FIG. 15. A good viscosity of printing ink is 10-12centipoises and a good surface tension of printing ink is 28-44dynes/cm, although other viscosities and surface tensions may beprovided in example embodiments.

Meniscus vacuum is a negative pressure for keeping the meniscus at theedge of nozzle. The user may change the value of meniscus vacuumdepending on the viscosity and surface tension of the ink. The typicalmeniscus vacuum value of water is 4 inches. If the meniscus vacuumnumber is not correct, the performance of ink may be affected with highfrequency.

The user may choose the number of nozzles from which to print, and thesystem may automatically compensate for the number of nozzles used butthe nozzles selected may only be one series of adjacent nozzles. In someimplementations, the printer has a drop watch camera which allows theuser to monitor, in real time, the drop performance. In this way, thecamera may assist the user to ensure that the nozzles chosen performanceuniformly.

Cleaning Cycles

In some examples, a cleaning cycle table allows the user to set nozzlecleaning processes before, during, and after printing. Setting cleaningcycle before printing may give a uniform start for every running of theprinter. Cleaning cycle may be especially important for some highviscosity applications, because setting cleaning cycle during printingmay prevent the high viscosity ink from clogging. Setting the cleaningcycle after printing may facilitate maintenance of the nozzles, as shownin FIG. 16.

Waveform

The printer software may have any suitable wavefore, for example, astandard 4-step waveform which is good for normal ink (e.g., ink havinga viscosity of 11-12 centipoises and a surface tension of 28-32dynes/cm. The 4 steps may include start, phase 1, phase 2, and phase 3.The basic concept for these 4 steps is use a bias voltage to controlpiezo actuation to suck a drop of ink and jet it with a controlledvelocity.

Waveform Start

In some examples, at a standby point, the nozzle voltage is set to a 40%level and is held for 1 μs. Under this condition, the channel which ispiezo basic slightly deflected and sucks some ink from cartridgestarting to eject, as shown in FIG. 17.

Waveform Phase 1

At phase 1, the voltage level may be set to 0 and held for, e.g., 3.584μs or any other suitable period, e.g., a period in a range from 3 μs to4 μs. The voltage brings the piezo back to a neutral straight positionwith the chamber at its maximum volume. In this phase, the fluid isfilled into chamber. FIG. 18 shows the meniscus at the nozzle edge.

Waveform Phase 2

Phase 2 is a firing pulse, as shown in FIG. 19. The steepness of theslope provides the energy for initial ejection and it is followed by ahold period. On the hold period, the voltage increases to 100% level andis held for, e.g., 3.712 μs or any other suitable period, e.g., a periodin a range from 3 μs to 4.5 μs. A this point, the chamber starts to jeta drop of ink. According to the hold time and voltage volume, thevelocity of a drop can be calculated.

Waveform Phase 3

Phase 3, the last phase of the waveform, is a return to standby, asshown in FIG. 20. First, the voltage level decreases to 70% and is holdit for, e.g., 3.392 μs (or any other suitable period, e.g., a period ina range from 3 μs to 4 μs), which is intended to prevent the printedhead from sucking air back in. Second, the voltage level is brought to40% level and chamber back to the standby position.

Those parameters mentioned above may play an important role duringprinting. They may advantageously be checked every printing runningaccording to the ink and material of substrate. Also ink condition mayslightly change according to room temperature and humidity level.

In further aspect of the invention, methods for printing of silk fibroininks using a inkjet printer are provided.

The prepared silk fibroin inks (functionalized or non-functionalized)can be filled into any types of liquid-refillable based cartridge forcommercial inkjet printers. The lifetime of the inks depends on theusage and the storage conditions. In some embodiments, storage in arefrigerator at 4 degree C. when finishing printing is recommended. Insome embodiments, silk inks (with our without dopants) may be storedwithout refrigeration, such as at room temperature (between about 18-26°C.) for an extended duration of time without significant loss offunction. High resolution (depending on the specific model of theprinter) can be achieved for printers with fine nozzle size and theaccess to control the nozzle operating performance (for example, firingvoltage and waveform, cleaning cycle, printing temperature and etc.)

Applications

The social benefits and commercial opportunities associated with theseand other systems define an important set of broader impacts. Thesustainable manufacturing approaches, the associated reduction inresource consumption and the decreased risk associated with chemical anddrug transport and handling constitute additional features with globalsignificance.

The development of every new generation of biomaterials has incorporatedefforts for the material properties to have an increasing degree ofinteraction with the biological environment. Biomaterials have evolvedfrom inert, monolithic matter, into “bio-instructive”, hybrid systems,with specific functionalities that are defined by correspondinglyspecific applications [1]. This approach limits the possibility to applya single biomaterial widely by favoring the functionality dictated tomeet stringent therapeutic requirements. Regulations that enforce theclinical use of biomaterials (e.g., FDA approval is not given to thebiomaterial per se but to its specific application) have alsocontributed to the current high specialization of biomaterials. Underthis approach, biomaterials are conceptualized as bio-high-techmaterials, which are provided to the end-user in their refined state, as“ready-for-use” products. The present application challenges thisviewpoint by developing the scientific underpinnings of a technologythat provides the end-user with a unified “base biomaterial” or platformmaterial, whose final utility can be easily and controllably defined atthe end-users' site.

In some embodiments of the invention described herein, the use of inkjetprinting (e.g., with personal printers) is provided. In someembodiments, inkjet printing of bio-inks (such as inks comprising astructural protein, e.g., silk fibroin and keratin) is provided as aplatform. In some embodiments, inkjet printing of bio-inks, such as silkfibroin inks, can enable the fabrication of custom printablebiomaterials for a variety of applications. In some embodiments, suchplatform is useful for optics, photonics, electronics, as well astherapeutics and sensing applications. Non-limiting examples of enabledapplications range from drug-doped printable inks that can preserveheat-sensitive biomacromolecules without refrigeration (elimination ofthe cold-chain), to functionalized silk ink libraries to be used forprintable drug formulation or activation (elimination of user-inducederror in drug reconstitution). Another concept involves biological andenvironmental analysis on orthogonal printing of ‘sensing’ silk inklibraries.

Work described in this application develops a scientific and engineeringbase of knowledge on the design of structural protein-based inks, bulkand surface properties, material-substrate interface, and manufacturingstrategies. An emphasis has been placed on the fundamental developmentof the working examples of particular types of bio-inks, including silkfibroin-based inkjet printing technology, as well as keratin basedinkjet printing technology. The targets in performance, reliability inoperation and scalability are comparable to those established incommercial inks for personal printers. As described in more detailherein, the present application provides working models of structuralprotein-based liquid compositions suitable for use as ink (e.g., paint,marker, and the like), including silk fibroin inks and keratin inks thatare suitable for use in conjunction with an inkjet printer. The work isa culmination of research and development efforts involving a deep andwide characterization of the effect of the printing process on thebiomaterials conducted, including the analysis of the fundamentals ofphase transformation incurred by the biomacromolecular inks during theirphase transformation in the printing process.

A set of device designs to address applications ranging from drugdelivery to biochemical analysis described herein is based at least onthe following: (1) development of bio-inks, such as silk inks, thatstabilize vaccines and integrate the vaccine reconstitution within theprinting process; (2) bio-inks that topographically control the releaseof antibiotics; (3) bio-inks that allows for basic biochemical analysisof biological samples, including human tissues (e.g. blood, urine); and,(4) bio-inks that spatially control stem cell fate by a controlledrelease of custom-printed growth factors.

Resorbable Silk-Based Electronics

The development of every new class of electronics has historicallyinvolved efforts to achieve operation in forms that undergo negligiblechange with time. More recently, with an opposite goal, novel silk-basedelectronics systems that physically disappear into the surroundingenvironment at prescribed times and rates have been described. The coreidea exploits transient electronics devices, which can be dissolved ondemand, upon the exposure to a specific stimulus. This research yieldedto the first biocompatible and bioresorbable electronic device designed,manufactured and implanted in vivo, which maintained its functionalitybefore being degraded by the body fluids. Here, the design of silkfibroin materials as substrate for the deposition of electronic circuitsplays a role as prominent as that in a fibroin-based tissue engineeredconstruct, where the control of scaffold degradation while exploitingits function is the main design criterion. The unique properties of silkfibroin, in fact, guarantee a broad processability portfolio togetherwith a fine-tuning of degradation kinetic both in vivo and in vitro. Theamino-acidic structure of fibroin well suites the possibility to beflexible, robust and well interfaced with materials such as metals,without negative phenomena such as delamination. These properties makethe resorbable electronic concept presently impossible to achieve usingany other material. With this background, silk fibroin ink compositionsand related methods described herein can be effectively employed inconjunction with the development and manufacture of transient electronicdevice described above.

Thus, example embodiments of the invention described herein combinediverse, but highly complementary, technology that will generatefoundational knowledge for both material design and material processing.New insights may be given in silk fibroin polymorphism, providing newapproaches for fibroin processing, fibroin phase transformation andfibroin interaction with several classes of biofunctionalmacromolecules. A new approach is needed for biomaterial design toenable a versatile technology that starts uses a common productionplatform (e.g., inkjet printing) yet covers a broad range of outcomesand applications. For this purpose, the standard processes of fibroinregeneration has to be re-invented and tuned to achieve usefuloperation; new processing approaches are essential for use withpractical inkjet systems through personal printers; new ways tostabilize fibroin solution are required to achieve useful operation.

Incorporating inkjet printing, bioinstructive biomacromolecules andbiomaterials into a single technology that offers high performance androbust operational characteristics has the potential to be agame-changer in the current biomaterials scenario. Many of theunderlying inventive concepts have applicability beyond the applicationexemplified in this application. For example, the inkjet printing offunctionalized silk fibroin-based inks will open the door tobiomaterial-based applications in energetics (printed silk-basedbatteries) and electronics (printed conductive silk). Indeed, the deepand extensive design, development and characterization of silk-based inkfor inkjet printing proposed in this program provides the route torobustly develop the aforementioned technology.

The stabilization of biomacromolecules in inkjet-printed biomaterials isan appealing concept that allows for the creation of structuralprotein-based ink libraries to print a variety of agents, such astherapeutics, sensing, and bioinstructive molecules. Interestingly, thecombination of the libraries may be left to the end-user, providingseveral degrees of freedom and a wide flexibility in the use of thetechnology described herein. Feedback from the end user will alsoincrease the portfolio of biomacromolecules incorporated and storedwithin the bio-inks, opening the possibility of applications such as aprintable pharmacy or biochemical lab. This has become possible, withthe development of a robust standard “biomaterial ink” for inkjetprinting technology as described herein.

Particularly compelling is the possibility to print stabilizedbiological agents, such as vaccines and antibiotics, by taking advantageof (a) the ability of silk to stabilize and preserve biological, and (b)the controllable transiency of fibroin and the consequent controlledrelease of such therapeutics. The former possibility addresses twoproblem of increasing urgency, driven by the high mortality in thedeveloping world from infectious diseases. In the recent years, theWorld Health Organization has underlined the urgency of addressing twopriorities: the cold chain system and the possible adverse eventfollowing immunization (AEFI).

Cold Chain System

More than 17 million people die every year from infectious diseases,particularly in the developing world. Vaccines and antibiotics areimportant components of an effective infectious disease containmentstrategy; antibiotics represent a rescue measure while vaccination canbe a primary mode of disease prevention. Unfortunately, the use ofvaccines and antibiotics is severely limited in the poorest countrieswhere infectious diseases account for more than half of all deaths. Dueto temperature sensitivity, vaccine and antibiotic formulations must bemaintained within a specific refrigeration temperature range. Becauseambient temperatures in the developing world deviate significantly fromrefrigeration temperatures, the successful delivery of active vaccinesand antibiotics depends on the cold chain system, a distribution networkto maintain optimal cold temperatures during transport, storage, andhandling. Cold chain requirements represent a major economic andlogistical burden, particularly in lower resource settings, whererefrigeration and electricity can be limited. The cold chain alone canaccount for 80% of the financial cost of vaccination and is estimated tocost vaccine programs $200-300 million per year. Deficiencies in theprocess frequently occur even in industrialized countries. Fortemperature sensitive compounds like vaccines and antibiotics,maintaining the cold chain is critical for adequate bioactivity.Failures in the cold chain result in costly waste and the loss of nearlyhalf of all global vaccines. Such failures can also result in thedelivery of ineffective, sub-therapeutic doses. For antibiotics, thisproblem can be associated with the development of antibiotic-resistantstrains, a major public health concern.

Adverse Event Following Immunization

An AEFI is any adverse event that follows immunization that is believedto be caused by the immunization. Immunization can produce adverseevents from the inherent properties of the vaccine (vaccine reaction),or some errors in the immunization process (programme error). Althoughrare in the developed countries, program errors are more frequent in thedeveloping world, where non-sufficient structures limit and negativelyinfluence the use of recommended immunization practices. Generally,these errors results from mistakes and/or accidents in vaccinepreparation, handling, or administration and results from the poorconditions in which immunization is practiced. Examples of the commonmistakes are inadequate shaking of the vaccine before use, errors in thereconstitution of vaccines before they are administered, contaminationof vaccine or injection equipment, use of a drug instead of a vaccine ordiluent, superficial injection, and use of frozen vaccine.

Current Applications of Bioprinting

Bio-printing allows for the dispensation of pico-sized quantities ofbiomaterial solutions (such as protein solution, nucleic acid solution,etc.) to designated sample space with minimal waste, both of which arehighly sought features in processing biological materials. What followsis a summary of exemplary applications, in which bio-printing isexplored.

Biosensors and Immunoassay Tests

From a life sciences/microelectromechanical systems (MEMS) standpoint,the ability to array proteins with good precision in uniform quantitiesis a useful feature both for the preparation of analytical samples, aswell as for the fabrication of devices. Protein microarrays allow forthe study of specific interactions between immobilized individualproteins and other biological systems. This ability translates directlyinto applications such as point-of-care clinical diagnostics andbiosensors. A biosensor is an analytical device that uses antibodies,enzymes, nucleic acids, microorganisms, isolated cells, or otherbiologically derived systems as a sensing element. In terms of cost,very small quantities of material are required to make sensors, andinkjet deposition lends itself well to mass production, allowing forsensors in many applications to be treated as disposable. The inherentlysmall size of the detectors involved implies the need for only smallquantities of material for analysis, resulting in greater sensitivity.Also, the need for a smaller amount of analyte offers faster detectorresponse times, since less time is required for the substrate tointeract with the analyte before a signal is detected. The use ofenzymes and antibodies as transducers also results in unmatchablespecificity for bioactive compounds. The limited stability of the manybiologically active materials involved limit the number ofmacromolecules that can be used to fabricate inkjet-printed biosensors.From a commercial standpoint, the areas where inkjet-printed biosensorshave gained the most interest has been disposable, point-of-carediagnostic products, such as glucose sensors, ELISA kits, analysis ofbiomarkers (particularly for detecting or monitoring cancers).

Inkjet Printing of Antibiotics and Growth Factors

Spatial control of bioactive molecule distribution in therapeutics andcell-stimulant molecules is highly sought. The ability to use UP tocontrol the topographical distribution of therapeutics may in fact leadto more effective and customized control of biofilm colonies formation,particularly in healing burns and in implantable prostheses. Theapplication of patterned growth factors on a cell-culture substrate(two- and three-dimensional) may lead to effective co-culture of cellsminimizing the problems of culture medium incompatibilities whileproviding bioinstructive persistent patterns. This would lead to thefunctional organization of multiple tissues types, addressing one of themajor bottlenecks in the engineering of the tissue-tissue interfaces(e.g. osteo-chondral and ligament-bone interface).

EXEMPLIFICATION

Although the present invention has been described with reference toparticular examples and embodiments, it should be understood that thepresent invention is not limited to those examples and embodiments.Moreover, the features of the particular examples and embodiments may beused in any combination. The present invention therefore includesvariations from the various examples and embodiments described herein,as will be apparent to one of skill in the art.

Example 1: Silk Fibroin Ink

Feasibility Demonstrations

Silk fibroin polymorphism overturns the general concept that structuralprotein with high molecular weight cannot be printed at highconcentration. Under certain conditions, regenerated silk fibroinpossesses the unique property of having a globular form. This allows forthe on-demand extrusion of the solution through nozzle with asub-micrometric diameter, mimicking the natural extrusion of silksolution. Unpublished work within our laboratory has developed aprotocol to obtain printable silk inks to establish a set of functionalsilk-based inks.

The following components has been used to explore the biomaterialparameter space and investigate the feasibility of a fibroin-basedbioprinting technology:

Printer

Dedicated (FujiFilm Dimatix Materials Printer DMP-2800) and personal(e.g. Epson Artisan 1430 and Epson WorkForce 30) printers have been usedto print functionalized fibroin-based inks. The dedicated printer hasbeen used to prove the concept of printing fibroin-based inks and toassess the different condition (e.g. process type, drop size) of theinkjet printing process. After a first screening, the piezoelectricdriven ejection of the drop has been preferred to the thermal one. Toobtain a robust method valid for different fibroin-based inks, a dropletsize in the range of 6-20 pl has been chosen. Within this range, infact, it was possible to successfully print all the fibroin-based inksinvestigated. Spurred by the results obtained with the dedicatedprinter, personal printers with specifications similar to theaforementioned values have been used for IJP of silk inks. Epson is theonly commercial brand to offer piezoelectric inkjet technology. Inaddition, Epson printers have a Variable Size Droplet Technology®, thatallows to choose between five pre-determined drop sizes (1.5 to 15 pl).CUPS drivers offered in Linux environment can be used to control thedroplet size through a 3 bit signal.

Substrate

The substrate is part and parcel of the IJP fibroin-based biomaterials.In accordance with the definition of hybrid and composite materials, theink-substrate system may be considered either a hybrid (no separateinterfaces between ink and substrate) or a composite (separateinterfaces). This distinction may strongly influence the structural,functional, mechanical and biological properties of the depositedproteins. In addition, different choices of substrates may tailor theproperties of the end product. Several materials are currently underinvestigation in our laboratories as silk-based UP substrates. Plainpaper is used both as a ‘standard’ substrate and as a substrate forcolorimetric sensing applications. Silk-based electrospun matrices areused for tissue engineering and therapeutics applications. Siliconwafers have been tested for microelectronics and biological assaysapplications. Tissue culture plastics are used for biologicalapplications. Microneedle-Silk-sheets may be used for vaccine delivery.

Silk Ink

We have established a robust protocol to print SF solution, bycontrolling the molecular weight (MW) of the protein through the fineregulation of the fibroin boil time (e.g. longer dissolution timecorresponds to a decrease in MW). A basic functional silk-based inklibrary has been explored by hybridizing regenerated silk solutions withdifferent dyes (a-c), antibiotics (e.g. penicillin), vaccines (e.g.Merck MMR II), photochromatic salt (e.g. AgCl, CuSO₄, CoCl₂),hygroscopic salts (MgCl₂), enzymes (e.g. glucose oxidase, pesin,Alcalase®, α-chymotrypsin). The functionality of inkjet-printedfibroin-penicillin hybrid has been recently tested and the antimicrobialefficacy of the antibiotic silk ink has been successfully assessed inpreliminary results (d).

Research on Bio-Printing of Functional Silk-Based Inks

We have developed an unprecedentedly versatile, inkjet printable,SF-based biomaterial. The possibility to ink-jet print SF may accompanythe SF-induced preservation, stabilization, and controlled-release ofbiomacromolecules with the possibility to obtain a programmabletopographic control of their release in the micro-scale. In addition, SFmay be successfully implemented to immobilize and stabilize sensingmolecules and to drive their interface with electronics device. SF hasbeen preferred to other abundant biopolymers (e.g. collagen, chitosanand keratin) due to the properties of the solvated protein. Whereas itis possible to regenerate SF in neutral aqueous solution atphysiologically relevant protein densities (1-15 wt %), soluble collagenis only obtainable at pH<4.0 (in the form of tropocollagen dimers ortrimers) and at low densities (<0.5 wt %), chitosan can only besolubilized in acidic conditions, and keratin is expensively obtained inaqueous solution only at low concentration (<0.5 wt %). Alternativecandidates as polycaprolactone, poly-hydroxyalkanoates, poly(lacticacid) (PLA), poly(glycolic acid) (PGA) and their co-polymer (PLGA) donot offer the polypeptidic nature of proteins, which highlights theopportunity offered by the unique properties of the silk biomaterial.Research involves an extensive characterization of neat and hybrid silksolutions (silk inks) to evaluate the interfaces between silk-silk andsilk-alto macromolecules, necessary both to fully understand thehybridization process and to control the UP process. In addition, theproject may be focused on protein characterization, IJP processingdesign and stability of ink-jet printed macromolecules. The envisionedoutcomes include a deep knowledge on the polymorphism of SF in thepresence of other macromolecules, both in liquid and solid phase. Thisfundamental material characterization will not only cope with thetechnological challenges involved in the IJP process of stablemacromolecules, but it will also open the door to their stabilization insilk solutions. This is a step towards therapeutic inkjet printingtechnology.

Silk Ink Design

Research on fibroin solution as a robust, printable biomaterial iscrucial to develop the SF-UP technology proposed here. Severalparameters influence the rheological and biochemical properties of SFsolutions, impacting the IJP process. In particular, a main focus of ourefforts has been to maximize the stability of the inks, withoutcompromising the achievement of a printable solution (i.e. particle size<200 nm, surface tension <65 mN/m, viscosity<10 mPa/s, pH=4-10).Although the project may leverage previous studies as a starting point,a new set of parameters other than MW may be used to control and tailorthe viscosity and the surface tension of the SF solution. pH, ionicstrength, protein and salts concentration may be considered in order totailor the viscosity and the surface tension of the silk solutions. Ingeneral, pH plays the major role in controlling the silk solution'sviscosity. A decrease in pH corresponds to a higher interaction betweenthe SF chains, particularly at values close to the isoelectric point(pH=4.8). In addition, for pH<3.5, the chains are reversibly aggregatingin crystalline β-sheet crystals, forming a gel structure. On the otherend, for strongly alkaline pH values (pH>9.5), SF chains irreversiblydegrade in small polypeptides. An optimum pH range between 6-8 may betherefore targeted in the design of the silk-based inks. Increasing saltconcentration in the SF solution increases its surface tension, which isalso dependent on the pH. The addition of small organic molecules may beused to decrease the surface tension of the SF solution. UnpublishedICP-MS measurements conducted on regenerated SF has shown the presenceof several cations, such as calcium, magnesium, iron(II) and copper(II).However, such ions are bound to the protein and are present only intraces. This is however an indication of the strong affinity of SF todivalent cations, which are been reported to have a strong stabilizationeffect on the molecules, with a consequent increase of viscosity atconcentrations ≥50 mM. Although the use of chelators to reduce the freedivalent cations is possible, it has to be considered thatmulti-carboxyl acids (e.g. EDTA, citric acids) are also reported tocross-link SF chains, producing an increase in the viscosity and in theparticle size. The combined effects of these parameters on the micellarand liquid crystalline form of SF chains in liquid phase may betherefore investigated. The morphological analysis of the SF solutionmay be accomplished with CryoTEM. The use of this technique to analyzeregenerated silk solutions may provide a snapshot of the chainconformation and aggregation within the solution in the differentconditions proposed. Circular dichroism (CD), dynamic light scattering(DLS), Raman spectroscopy and attenuated total reflectance Fouriertransform infrared (ATR-FTIR) spectroscopy may be used to monitor theeffects of the aforementioned parameters on SF chain distribution,profile, orientation and structure. Although some studies alreadydescribed the effect of pH, ionic strength and salt concentration on SFregenerated solution properties, there is a lack of knowledge of thecombined effects of these three parameters and their mutualinterdependence. Viscosity and surface tension measurements may evaluatethe efficacy of the proposed changes. With the exception of CryoTEM, allthe instruments necessary to achieve a deep characterization of SFsolution are available in house. Transmission electron microscopy may becarried out at the NSF-funded Harvard Center for Nanoscale System (CNS),as for previous studies.

Device Design

The above-described fundamental characterization and optimization offibroin solution for IJP may build the necessary know-how to cope withthe difficulties that occur during hybridization of SF withbiofunctional macromolecules to be used as therapeutics (i.e.,antibiotics, vaccines) or sensors (glucose oxidase, hemoglobin).Although the incorporation of a large variety of macromolecules in SFsolution has been previously reported, little is known onsilk-biomacromolecule interactions. The latter may affect thetherapeutic compound's stability and functionality, silk structure, andrheological properties of the solution. Indeed these aspects are crucialfor the scope of the work and may be investigated during the course ofthis work.

Based on our previous work, we are able to pursue hybridized silk inkswith antibiotics (e.g., penicillin, neomycin, gentamycin, streptomycin,and tetracycline), vaccines (e.g., Merck® M-M-R® II—Measles, Mumps, andRubella Virus Vaccine), enzymes (glucose oxidase) and growth factors (tocontrol human mesenchymal stem cells (h-MSCs) fate toward osteogenic(ascorbic acid, 3-glycerolphosphate and dexamethasone) and chondrogenic(IGF-1, TGF-β) lineages. Such hybridized silk inks may be characterizedat three different levels by evaluating the effects of the hybridizationon (1) rheological properties of the solution (2) SF chains structureand aggregation, and (3) preservation and functionality of thebiofunctional macromolecules incorporated. Successful hybridization maysatisfy the criteria of printability while preserving the functionalityof the macromolecules incorporated.

The problem may be approached by hybridizing different amounts of any ofthe above mentioned macromolecule with the current fibroin standardsolution for printing. The analysis of the rheological properties of thesolution together with the measurement of the particle size may thenprovide an indication of the printability of the hybrid inks. If therequirements to obtain a printable solution are not met (particularlydue to the aggregation of SF chain in the solution to formparticles >200 nm), strategies such as the use of additives (e.g.,divalent cations), changes in pH, or a reduction in SF concentration maybe used, in accordance with the know-how acquired during silk inkdevelopment (Paragraph 6.1). The effects of hybridization on thestructure of SF in solution may also be characterized with thepreviously described techniques (FTIR and Raman spectroscopy, CD, DLS).Specific assays may be used to evaluate the stability of the differentbiofunctional macromolecules in the aqueous SF environment beforebio-printing, in accordance with previously developed and adoptedprotocols. More specifically:

Vaccines: Two distinct studies have been conducted. i) Stabilization ofvaccine in SF solution—This is a fundamental study to investigate the SFchains-vaccine interaction in solution over a prolonged exposure time.The study is preparatory for device-oriented application. SF solutionhybridized with increasing amount of reconstituted vaccine may be storedat 4° C., 25° C., and 37° C. for 1, 3, and 7, 14 and 28 days. Thestabilization of the trivalent vaccine investigated may be accomplishedas regulated by the World Health Organization (WHO): 1) the vaccineshould retain at least 1,000 live virus particles in each human doseafter incubation at 37° C. for 7 days and 2) virus titer should notdecrease by more than 1 log₁₀ during storage. Viral infectivity assaymay be used to evaluate vaccine stabilization, by employing RT-PCR, asrecently published. As made, printed trivalent vaccine solutions withand without SF may be used as positive controls. ii) printed silk inksthat stabilize vaccines and integrate the vaccine reconstitution withinthe printing process. This is a device-oriented study. As madeSF-vaccine solutions with increasing concentrations of vaccine may beprinted on different substrates (e.g. plain paper, TCP, andmicroneedle-silk substrates). The so formed biomaterials may be exposedto increasing temperatures (4° C., 25° C., and 37° C. for 1, 3, and 7,14 and 28 days). Upon exposure to these conditions, avaccine-reconstitution ink will then be printed on top of the printedsilk-vaccine layers and the aforementioned viral infectivity assay maythen be used to evaluate vaccine stabilization. This experiment maycover an interesting future scenario given that the molecules thatcompose the reconstitution ink are stable in water and may therefore beshipped included in a commercially available inkjet cartridge compatiblewith most of personal printers and may be subsequently used in situ toreconstitute pre-printed vaccines.Sensor: Glucose oxidase, a flavin adenine dinucleotide dependent enzyme(GOD-FAD), may be used to manufacture silk-based glucose sensors. Theenzyme shows high substrate specificity for β-d-glucose oxidation withmolecular oxygen:β-d-glucose+GOD-FAD

GOD-FAD.H₂+δ-d-gluconolactoneGOD-FAD·H₂→GOD-FAD+H₂O₂β-d-glucose is oxidized to δ-gluconolactone with concomitant reductionof GOD-FAD. The reduced form of GOD-FAD is regenerated to its oxidizedform by molecular oxygen to produce hydrogen peroxide.

Two different sensors have been investigated by exposing the printedbiomaterials to known concentration of glucose. First, differentconcentrations of glucose oxidase (derived from Aspergillus niger) canbe incorporated in SF solutions and printed on electrodes built onsilicon wafers to form sensor arrays. SF has already shown to provide astructural and functional role in interfacing the biological milieu withelectrodes and may also serve to immobilize the enzyme. The electricsignal generated from the reaction can be monitored by any suitablemeans.

Secondly, two inks can be used simultaneously. SF-glucose oxidase inkmay be co-printed with a silk ink hybridized with peroxidase and achromogenic system (3,5-dichloro-2-hydroxybenzenesulfonicacid/4-aminoantipyrine). After exposure to glucose, the hydrogenperoxide generated from glucose oxidase reduction may react with thechromogenic system (reaction catalyzed by peroxidase), to generate apink colorimetric response (intensity as a function of glucoseconcentration), with an peak absorption at λ=514 nm. The stability ofthe proposed hybrid solutions may be evaluated as made and after storingthem for 1, 3 and 7 days at 4° C. and 22° C. A commercial colorimetricglucose sensor may be used as control.

Antibiotics: SF solution hybridized with increasing amount ofantibiotics may be stored at 4° C., 25° C., 37° C. and 45° C. for 0, 1,3, 7, 14 and 28 days (tetracyclines may be protected for the whole timefrom light exposure). Stabilization may be evaluated by printing apattern with a gradient of antibiotics on tissue culture plastic (TCP)with previously cultured bacteria strains (e.g., E. Coli ATCC 25922 andS. Aureus ATCC 2593-cultured for 18-24 hours, to an optical density(OD600) between 0.8 and 1 or equivalent to 107-108 CFU/ml).Effectiveness in control of the topographical-selective destruction ofbacterial culture may be evaluated by measuring zones of clearance, whencompared with positive controls (inkjet-printed fresh antibiotic hybridinks and antibiotic solutions (without SF) with known concentration ofactive therapeutic). In addition, as made SF solution hybridized withantibiotics may be printed with a controlled pattern and a biologicalgradient on gauzes, while bacteria strains may be cultured on a TCP aspreviously described. The printed gauzes may be then applied to thebacterial cultures and the effectiveness of antibacterial activity maybe evaluated by measuring the zones of clearance, when compared to apositive controls (antibiotic solution inkjet printed on gauzes).Growth factors: Different concentrations of osteogenic and chondrogenicgrowth factors (GFs) were incorporated in SF solution and printed on TCPand on an electrospun silk fibroin (ES-SF) matrices. The printedsilk-based biomaterial is water annealed for 6, 12 and 24 hours toinduce an increased amount of crystallinity in SF, which corresponds toa slower release of GFs. As preliminary study, the release of GFs incell culture medium in the presence of physiologically relevantconcentration of MMPs were evaluated with specific immunoassays. h-MSCsresponse to GFs release may be evaluated at days 7, 14 and 21 withRT-PCR. In particular, the total RNA may be extracted from h-MSC-seededscaffolds with TRIzol® (Invitrogen, Carlsbad, Calif., USA) following themanufacturer's instructions. After RNA separation, nucleic acidconcentration and integrity may be determined with an EppendorfBioPhotometer Plus (Eppendorf, Hamburg, Germany). 250 ng of total RNAmay be mixed with 0.25 ng of random hexamers (Invitrogen, Carlsbad,Calif., USA) and reverse-transcribed into complementary DNA (cDNA) with200 U of Superscript Reverse Transcriptase II (Invitrogen, Carlsbad,Calif.) and RNasin (Promega). Quantitative RT-PCR may be performed withan ABI Prism 7900 HT 139 (Applied Biosystems). Each PCR reaction maycontain 9 μl of cDNA, 0.5 μl of both forward and reverse primers (10μM), and 10 μl of SYBR Green (Applied Biosystems). The cyclingconditions may be, for example: 50° C. for 2 minutes, initialdenaturation at 95° C. for 10 minutes, and 45 cycles of 15 seconds at95° C. and 1 minute at 60° C. Relative quantification of target geneexpression may be achieved first normalizing to an endogenous referencegene (housekeeping gene GAPDH) to correct different amounts of inputRNA, and then relating the expression of the target genes to a referencesample (h-MSCs extracted from relative control without GFs) using the−2ΔΔCt method. h-MSC cultured with standard procedures on TCP may beused as control.Silk Ink—Substrate Interfaces

As previously stated, the substrate is a vital component of IJPfibroin-based biomaterials. The deposition of silk inks on the substratemay be affected by adsorption, which affects the structural, functional,mechanical and biological properties of the deposited proteins. Inaddition, different choices of substrate may influence the properties ofthe biomaterials, bringing unique features to the end product. Severalmaterials are currently under further investigation as silk-based IJPsubstrates in our laboratory. Among these, are plain paper (used both asa ‘standard’ substrate and for colorimetric sensing applications),silk-microneedle sheets (for therapeutic and vaccine delivery),silk-based electrospun mats (used for tissue engineering andtherapeutics applications), silicon wafers (for microelectronics andbiological assays applications), and TCP (for biological applications).

The morphology of the ink-substrate interfaces for all theaforementioned substrates (diagnostics are available in-house) preparedin accordance with the present application can be evaluated usingsuitable techniques, such as through scanning electron microscopy (SEM),and atomic force microscopy (AFM). In addition, detachment forcemeasurements at the silk ink/substrate interface for differentcrystallinity of SF-based inks may be investigated through AFMmeasurements, as previously reported. Characterization of the printedfibroin structure on different substrates may be studied with FTIR andRaman spectroscopy for both neat SF and hybrid silk inks.

Example 2: Keratin Ink

Referring to FIGS. 55 to 59, a keratin-based ink was prepared using woolas a starting material. To prepare delipided wool, the followingprotocol was used.

Australian Merino 64's top wool (30 g) was rinse in distilled water (6liters, 30° C.) for 30 minutes (three water changes every 10 minutes),blotted and dried under vacuum for 6 hours. Lipid extraction was thenperformed with 100% acetone for 24 hours to remove remaining unboundsurface lipids. The fibers were then washed (3×) with distilled water (6liters, 30° C.) for 3 hours (three water changes every 45 min) andair-dried.

The delipided wool fibers were then cut into short fibers, 3 mm long. Amixture of 7M urea (500 ml), 2-mercaptoethanol (50 ml) and 0.5Mthiolurea (50 ml), was used to solubilized 30 g of keratin at roomtemperature for 24 h. The solution was then filtered through a stainlesssteel sieve (#200) and dialyzed in dialysis cassettes (3,500 MW cut-off)against distilled water (8 liters) for 72 hours (changed every 6 hours).The so obtained keratin solution was then centrifuged twice (5° C., 9000rpm, 20 min per cycle). The regenerated keratin solution was thenconcentrated to 7 wt % (70 mg/ml) through centrifugation in vacuum.

The so obtained regenerated ink was then used for ink jet printing asdescribed before with silk fibroin.

It was observed that the solution of keratin at 6 wt % has substantiallythe same rheological properties of the one of silk fibroin at the sameconcentration. The MW of the so obtained keratin is around 40-60 kDawith a second band at 15-20 kDa.

Example 3: Demonstration and Characterization of Silk Fibroin Ink andits Utility in Inkjet Printing

(The accompanying document describing this portion of work isincorporated herein by reference in its entirety.)

Project Report: Directly Printing of Silk Fibroin Based Patterns andDevices Using a Commercial Inkjet Printer Fujifilm Dimatix™ DMP 2800Printer

1 INTRODUCTION AND BACKGROUND

Silk is generally considered as a protein polymer that are usuallysynthesized within specialized glands and then spun into fibers by someLepidoptera larvae such as spiders, silkworms, mites and flies [1-3].Silks generated by different species differ widely in composition,structure and their mechanical and chemical properties. In this work,efforts will be focused on the silk generated from the domesticatedsilkworm, i.e. Bombyx mori, partially due to the convenience to the silksource and the extensive experience and knowledge that the Department ofBiomedical Engineering at Tufts University has gained in the past twodecades.

It is worth mentioning that each of the different silks has a differentamino acid composition that further determines the mechanical propertiesfor their specific functions and the forms of the end product such asreproduction as silk cocoons, silk webs, and etc. Even within the “same”type of larvae—for example Bombyx mori silkworms—the silkworms fromdifferent locations slightly differ. In this work, we mainly discuss thesilk fibroin regenerated from the silk cocoons from Japan.

The first use of the silkworm silk for biomedical purposes can be chasedback to centuries ago (mainly for silk sutures for wound closure).However, it was found raw silk fibers cause an inflammatory responsethat was later found to be due to the sericin (a “glue”-like proteinthat holds silk fibroins to form the cocoon). After careful removal ofthe sericin [4], the purified silk proteins have been used in manybiomedical applications and have proven to be effective in many clinicalapplications [5].

1.1 Biocompatibility of Silk

Silkworm silk fibers have been used in biomedical applications,particularly as sutures for wound ligation. However, some biologicalresponses to the protein were found, which raised questions about thebiocompatibility of the protein [6-8]. There have been some difficultiesin identifying the exact source that causes the biological responses dueto the lack of detailed characterization of the silk fibers preparedunder different conditions. The silk from Bombyx mori silkworms containsat least two main silk fibroin proteins (i.e. light and heavy chainswith the molecular weight of ˜25 and 325 kDa, respectively) that areencased in a coat of sericin. Later on, it is clear that—based on manystudies [9-10]—the sericin glue-like proteins are the major cause ofadverse reactions while the core silk fibroin fibers appear to be quitebiocompatible and to be comparable to most other commonly usedbiomaterials [11]. The biocompatibility was evaluated for humanmesenchymal stem cells (hMSC) and the inflammatory responses wereconsiderably lower in cells cultured on silk fibroin films as comparedto collagen and poly(lactic acid) films [12].

1.2 Structure and Properties of Silk

A continuous silk thread (with a length over 1 kilometer) can be drawnfrom a single silk cocoon. The fibroin is a huge molecule consisting ofboth amorphous region (˜1/3, commonly termed as Silk I) and crystallineportion (˜2/3, Silk II), as shown in Table 3. Silk I is a water-solublestructure while Silk II excludes waters and is therefore insoluble inwater and some other mild acid and alkaline solutions [13].

TABLE 3 Structure of silk fibers [13]. Bombyx mori silk worm Silk fiberSilk fibroin (72-81%) P 25 Silk sericin (19-58%) H chain L chainglycoprotein a glue-like protein Molecular 325 kDa 25 kDa 25 kDa ~380kDa Weight Polarity Hydrophilic Hydrophilic Structure silk I(random-coilor unordered non-crystalline structure) structure silk II(crystallinestructure) silk III (unstable structure) Function the structure proteinof fibers binds two fibroins filament core protein together coatingprotein

The crystalline portion contains repetitive amino acids along itssequence (-Gly-Al-Gly-Ala-Gly-Ser—), resulting in an antiparallel betasheet. And it is this beta sheet structure that leads to theextraordinary stability and mechanical properties of the fiber such asremarkable strength and toughness. The toughness of silk fibers is foundto be even greater than the best synthetic materials, including Kevlar[14]. And in terms of strength, silk is significantly superior to mostof commonly used polymeric biodegradable biomaterials such as collagen.A comparison of the mechanical properties of silk and otherbiodegradable materials is shown in Table 4.

TABLE 4 Mechanical properties of biodegradable materials [13]. Source ofUTS Modulus Strain (%) biomaterial (MPa) (GPa) at breakage Bombyx morisilk (with 500 5-12 19 sericin) Bombyx mori silk 610-690 15-17  4-16(without sericin) Bombyx mori silk 740 10 20 Collagen 0.9-7.40.0018-0.046  24-68 Cross-linked collagen 47-72 0.4-0.8 12-16 Polylacticacid 28-50 1.2-3.0 2-61.3 Regeneration of Silk Protein from Bombyx mori Silk Fibers

Silk has been processed to a range of biomaterials such as films, gels,fibers and sponges. The starting point of those silk-based materials isthe regeneration (alternatively called extraction) of silk fibroinprotein from silk fibers. In this section, the regeneration of silkprotein (for printable silk ink grade) from Bombyx mori silkfibers/cocoons is shown in FIG. 3 and briefly described as followings:

-   1 Fill a 2 liter glass beaker filled with 2 liters of ultrapure    water and heat until boiling;-   2 Weigh 4.24 grams of sodium carbonate (Na₂CO₃) and add it into 2    liters of ultrapure water prepared in Step 1 (to prepare a 0.02 M    solution);-   3 Prepare and weigh 5 grams of silk fiber or pre-cut silk cocoon    pieces and add it into the boiled Na₂CO₃ solution;-   4 Boil for 120 min with occasionally stirring;-   5 Remove the silk fibroin with a colander and rinse it in ultrapure    cold water;-   6 Rinse the fibroin in ultrapure water for 20 min;-   7 Change the water and repeat step 6 for twice (for a total of three    rinses);-   8 Remove the silk and then spread it out to dry in a fume hood for    24 hours;-   9 Prepare a 9.3 M lithium bromide (LiBr) solution (the volume of    LiBr solution equals four times of the weight of the dried silk in    gram). For example, for 1 gram of dry silk processed by Step 1-8,    prepare 4 mL LiBr solution;-   10 Pack silk fibroin tightly into a glass beaker and add the    required amount of LiBr solution calculated in Step 9 on top;-   11 Put the beaker in an oven at 60 degree C. for at least 4 hour;-   12 Insert the dissolved silk (in LiBr solution) solution into    dialysis cassette for 48 hours. Change the water every 8 hours (for    a total of 6 changes);-   13 Centrifuge to remove impurities. Spin rate: 9,000 r.p.m. (i.e.    ˜12,700 g) at 4 degree C. for 1 hour;-   14 Store the purified silk solution in a fridge at 4 degree C.

The entire extraction process takes about 4 days. The resulted silksolution can be used for printing as it is or can be doped withappropriate dopants for specific applications.

1.4 Material Formats and Fabrication Methods of Silk Fibroin Protein

The extracted silk fibroin can be further processed into differentmaterial formats for a range of potential applications, as shown in FIG.2 and Table 5.

The fabrication methods of some popular silk materials are brieflylisted as followings [1]:

Silk gels: One important material option for silk-based biomaterials isthe formation of hydrogels. Silk hydrogels can be formed throughvortexing (without needing to contact the solution with a probe),sonication (a simple method to produce silk gels), and the applicationof electrical current (the gelation process is reversible by reversingthe polarity of the control voltage).Silk films: Among those material forms, silk films are of particularinterest for bio-optics and bio-photonics applications due to theiroutstanding transparency and surface smoothness. Silk films can bereadily fabricated using both spin-coating process (for extremely thinfilms with the thickness ranging from a few nanometers to submicron; thethickness can be controlled by adjusting the concentration of the silksolution and spin-coating rate.) and soft-lithograph-like castingprocess (for films with the thickness of a few microns to hundreds ofmicrons). Furthermore pouring the silk fibroin solution on to apre-patterned substrate can reproduce silk films that replicate thepatterns on the substrate.

TABLE 5 Biomedical applications of various silk material formats [15].Application Tissue type Material format Tissue engineering Bone HFIPsponges^(41,43,44) Aqueous sponges^(43,45,46) Electrosoun fibers³⁶Cartilage HFIP sponges⁴⁷ Aqueous sponges⁴⁸⁻⁵⁰ Electrospun fibers⁵¹ Softtissue HFIP sponges⁵² Aqueous sponges⁵² Hydrogels¹⁵ Corneal tissuePatterned silk fitrns^(31,53) Vascular tissues Tubes²² Electrospunfibers⁵⁴⁻⁵⁶ Cervical tissue Aqueous sponges⁵⁷ Skin Electrospunfibers^(56,59) Disease models Breast cancer HFIP sponges⁶⁰ Aqueoussponges^(61,62) Autosomal dominant Aqueous sponges⁶³ polycystic kidneydisease Implant devices Anterior cruciate ligament Fibers^(54,65) Femurdefects HFIP sponges¹⁷ Mandibular defects Aqueous sponges^(65,67) Drugdelivery Drug delivery Spheres^(34,58-70) Growth factor deliverySpheres⁷¹ Small molecule Spheres³³

Silk sponges: Biomaterials play a key role in tissue engineering. Silksponges (as a versatile 3D porous scaffolding material) allow cellsseeded within or on the matrix and have the advantage of being able todegrade into biocompatible fragments afterwards. Silk fibroin offersversatility in terms of matrix design for a number of tissue engineeringneeds. Aqueous based porous silk sponges can be fabricated usingvariable size salt crystals as porogen and manipulating theconcentration of silk solution and the salt size.

Silk fibers: Silk fibers (for silk mats) are of particular interest asbiomaterials due to the increased surface area and rougher topographythat facilitate cell attachment. Silk fibers can be prepared by directlydrawing the fibers from silk solution or by electrospinning. Silk fiberscan be produced in a wide range of diameters (ranging from a fewnanometers to tens of microns).

1.5 Inkjet Printing Technique and FUJIFILM DMP Series Printers

It is almost a “no-brainer” to come up with the idea of turning the silksolution into the “ink” for directly printing silk devices using asuitable printer. It is very challenging though since there are certainrequirements on the silk as ink. Inkjet printers have grown inpopularity and performance—actually, inkjet printers are by far the mostpopular—since their introduction in the latter half of the 1980s.Compared to laser printers (that use dry ink, also known as toner,static electricity and heat to print), inkjet printers use liquid inksand nozzles (usually multiple nozzles needed) to spray drops of inkdirectly onto the substrates.

A typical inkjet printer includes: a) print head—that contains a seriesof nozzles that are used to spray the ink drops; b) ink cartridge—thatcontains the ink; c) stepper motor—that moves the print head back andforth across the substrate.

It is worth mentioning that nowadays most inkjet printers usepiezoelectric nozzle technique for precision printing, which use piezocrystals that vibrate when receive a tiny electric charge. When thecrystal vibrates inward and out ward, it pulls and forces a tiny amountof ink and sprays it out of the nozzle.

In this work, we use a commercial inkjet printer from FUJIFILM, DimatixMaterials Printer DMP-2800, as shown in FIG. 6. It uses piezoelectricinkjet technology and MEMS fabrication processes (for cartridges,nozzles and etc.).

As shown in FIG. 7, the DMP-2800 series printer works with a maximumprintable area of A4 size substrate (8×11 inch) with a disposable (butreusable with certain modifications/tweaks) piezo inkjet cartridge. Themaximum height of printable substrate is up to 25 mm. It also has theability to heat up the substrate up to 60 degree C. In addition, thereis a fiducially camera available allowing real time watching theformation of the drop as it is ejected from the nozzle.

The Dimatix Materials Desktop Printer DMP 2800 is a convenientlaboratory tool that enables users (i.e. students, researchers andengineers) to evaluate the use of specific ink (i.e. silk fibroinsolution in our case) for new and proof of principle technologydevelopment with extensive flexibilities to optimize process parametersfor user oriented applications.

In the rest of this section, several typical applications of using DMP2800 and various types of functional materials and printable inks willbe listed.

Printer-compatible substrates:

Paper, Kapton (i.e. polyimide), poyethylene (PET), fabrics (such ascotton, nylon, polyester), metals (such as aluminum foil, copper foil,stainless steel foil and etc.), liquid crystal polymer, palladium, andglass.

Printer-compatible fluids/inks:

Conductive silvers, conductive inorganics (non silver ink, such as ITOinks), conductive organics (such as OLED), single wall carbon nanotubes(SWCNTs), insulators, polyimides, photoresists, resins, and UV curableinks.

For different types of conductive inks, they usually have a wide rangeof ink properties including viscosity, density, surface tension, anddispersion stability. Therefore, it is necessary to optimize the printerparameters such as the volume of the jetted ink, the gap distancebetween droplets, the printing frequency, temperatures of the jetted inkand the substrate, and the sintering/curing mechanism performed afterprinting. One of the most popular applications of using inkjet printerfor conductive printing is rapid printing of RFID tags. However, it is arather challenging endeavor since precise control of the desiredconductivity and pattern designs (on non-perfect substrates, forexample, on non-glossy rough papers).

Unlike the traditional photolithograph and etching PCB fabricationprocess (which is a subtractive method by removing undesired metal fromthe substrate surface), conductive inkjet printing for RF applicationsjets the single conductive ink droplet from the nozzle to pre-definedposition (usually controlled by computer and a precise motorstepper)—therefore, no harsh chemicals as the etching wastecreated-resulting in an economical and ecological fabrication solution.Silver nano-particle inks are and usually selected and commonly used forgood metal conductivity. As mentioned above, a sintering process eitherby applying heat or UV exposure (to remove excess solvent and to removematerial impurities) is usually needed, which also enhance the bondstrength between the ink and the as-printed substrate. Note that animmediate sintering process is essential, because the silk ink begins tooxidize that would render the conductivity of efficiency of the metallicpatterns.

As shown in FIG. 8, there are gaps between printed silver nanoparticlesafter printing, resulting in a poor connection and therefore notconductive. After 15 minutes of heating/curing process, the particlesbegin to aggregate and gaps start to diminish, which forms a continuousmetal film and guarantees a good conductor, which determines theperformance of the printed electric devices (e.g. RFID tags, as shown inFIG. 9 and FIG. 10).

Another arena of inkjet printing technique is so called “bioprinting”,which requires micro level (and in many cases, nano level) liquidmanipulation. The typical applications include micro-dosing, biochemicalsurface patterning and modification, tissue engineering and importantlythe direct placement of living cells, DNA arrays, and proteomics [19].Questions have been raised on the influence of mechanical forces andrelatively intense electric field during the inkjet process on the cellsand some research reports showed that although some cell death mayoccur, surviving cells recover rapidly and seem to behave normally, asshown in FIG. 11.

2 WORKING PRINCIPLE OF DIMATIX MATERIALS PRINTER

We use DMP-2800 inkjet printer from FUJIFILM Dimatix, Inc to directlyprint regenerated silk fibroin protein. The DMP-2800 printer can createyour own patterns and load BMP patterns over an area of about 200*300mm. The printer allows the substrates up to 25 mm thick with anadjustable Z height. It also includes 16 piezo-based jetting nozzles at254 μm spacing and a fillable cartridge. In this way, we invent a methodto regenerate silk fibroin protein as the ink to match specific fluidrequirement of this printer. The printed silk protein is biocompatibleand can be further functionalized by mixing the silk ink withappropriate dopants (including both organic and inorganic ones) forspecific applications.

2.1 Dimatix Materials Printer 2800 Operation

The DMP-2800 is a powerful laboratory tool which has capabilities toallow user to optimize process parameters, like nozzle voltage,substrate height, and wave form. Different from other commercialprinter, DMP-2800 provides multi-layer printing and allows alignmentprocess when using multiple cartridges and matching the origin point onthe substrate. To brief introduce the operation of the printer, theprocess includes creating pattern, loading ink, setting printingpermanents.

2.1.1 Create Pattern

The DMP software only accepts DMP files or let you create patterns usingPattern Editor. Pattern Editor allows you to modify pattern of drops forprinting, so it is good for some fine and small scale patterns, forexample, patterns from FIG. 12. However, it takes a long time to createa complex structure pattern for printing. Patterns like FIG. 13, youneed transform to BMP file first, and then import them to DMP software.

2.1.1.1 BMP Pattern Import

To print a fine pattern, first make a high resolution original filebefore you transform it to BMP file, because the DMP soft ware onlyallow you import an monochrome Bitmap file which is quite low resolutionfile. After creating Bitmap pattern, selecting Pattern Editor (Bitmapimages) from the DDM main window and choosing a drop space size whichdepends on your ink and substrate before importing the Bitmap file.Double check the final size of the pattern. If the final size is notyour exception, you can adjust the final size by changing the patternsize in Bitmap file, then reloading your pattern. And repeat yourpattern by controlling the placement number.

2.1.1.2 Create User's Own Patterns

Select Tools on the DDM main window, click Pattern Edition. It willallows you to create your pattern by enter dimension on Pattern BlockDrop Positions. On the other way, you can draw a feature through PreviewDrops Window. Before you create your pattern, you still need choose thesize of Drop Spacing which is the center to center distance from onedrop to the next. For a normal silk drop is about 25 μm on hydrophilicsurface, like silicon wafer.

2.1.2 Loading Cartridge

The ink which you fill into the cartridge need go through 0.22 μm filterfirst, because the size of a nozzle is 26 μm and the volume of a drop is10 pL. It means the nozzle is easy clogged by particle size larger than0.22 m. After loading new cartridge onto the printer, change the cleanpad for new ink due to not contaminating by other chemicals. Afterloading your cartridge, select the pattern you want to print from theSelect Pattern. The system will automatic calculate the CartridgeMounting Angle determined by drop spacing specified in the pattern. Hereis a table (Table 2) to compare the relation of saber angle, resolution,and drop spacing.

2.1.3 Setting Parameter for Printing

Click the Drop Watch button, the system will move the cartridge to theright side of the platen, positioning the nozzles over the drop watchercamera system. First, select the range of nozzles you wish to jet yourpattern. Second, modify the nozzles to performance uniform by adjustingvoltage of those nozzle monitored by camera. Another important parametershould be set up carefully is the cartridge print height according tothe substrate thickness. If the substrate thickness is less than 0.5 mm,the printable range is 210 mm*315 mm. If the substrate thickness isbetween 0.5 mm to 25 mm, the printable range is 210 mm*260 mm. And therepeatability distance is ±25 μm. The last step before printing isalignment process through Fiducial Camera from the tools menu on the DDMwindow. First, calibrate the position of a new cartridge or head angleby setting the Drop Offset automatic or manual from tools menu. Second,set the printing origin point and reference point for multiple layersprinting.

TABLE 2 Resolutions relationships Saber Drop Resolution angle spacing[dpi] [°] [μm] 5080.00 1.1 5 2540 2.3 10 1693.33 3.4 15 1270.00 4.5 201016.00 5.6 25 846.67 6.8 30 725.71 7.9 35 635.00 9.1 40 564.44 10.2 45508.00 11.4 50 461.82 12.5 55 423.33 13.7 60 390.77 14.8 65 362.86 16.070 338.67 17.2 75 317.50 18.4 80 298.82 19.6 85 282.22 20.8 90 267.3722.0 95 254.00 23.2 100 241.90 24.4 105 230.91 25.7 110 220.87 26.9 115211.67 28.2 120 203.20 29.5 125 195.38 30.8 130 188.15 32.1 135 181.4333.4 140 175.17 34.8 145 169.33 36.2 150 163.87 37.6 155 158.75 39.0 160153.94 40.5 165 149.41 42.0 170 145.41 43.5 175 141.11 45.1 180 137.3046.7 185 133.68 48.4 190 130.26 50.1 195 127.00 51.9 200 123.90 53.8 205120.95 55.8 210 118.14 57.8 215 115.45 60.0 220 112.89 62.4 225 110.4364.9 230 108.09 67.7 235 105.83 70.9 240 103.67 74.7 245 101.60 79.8 250100.00 90 2542.2 Cartridge Parameter Setting

To optimize drop performance, there are some parameters need be set upprecisely. The main parameter includes nozzles voltage, nozzletemperature, meniscus set point, cleaning cycles, and waveform.

2.2.1 Nozzles Voltage

Clicking on the Edition button the cartridge setting box, the followscreen displays. The voltage of each nozzle can be individual adjusted,as shown in FIG. 14. Increasing voltage will increase drop volume andjetting velocity. And a good velocity to be set is 7-9 m/sec.

2.2.2 Nozzles Temperature, Meniscus Set Point, Nozzle Number, PrintHeight

Clicking on the cartridge tab in the Cartridge Settings, you can adjustnozzles temperature, meniscus set point, nozzle number and print height.

We can lower the ink viscosity and surface tension by increasing nozzletemperature. The printer allows you to adjust nozzle temperature from 28degree C. to 70 degree C., as shown in FIG. 15. A good viscosity ofprinting ink is 10-12 centipoises and a good surface tension of printingink is 28-44 dynes/cm.

Meniscus Vacuum is a negative pressure for keeping the meniscus at theedge of nozzle. Change the value of meniscus vacuum depends on theviscosity and surface tension of the ink. The typical meniscus vacuumvalue of water is 4 inches. If the meniscus vacuum number is notcorrect, it would affect the performance of ink with high frequency.

Choose the number of nozzles to print, and the system will automaticallycompensates for the number of nozzles used but the nozzles selected canonly be one series of adjacent nozzles. The printer has drop watchcamera which allows you to real time monitor drop performance. In thisway, the camera will help you to make sure the nozzles you chooseperformance uniform.

2.2.3 Cleaning Cycles

The cleaning cycle table lets you setting nozzles cleaning processbefore, during and after printing. Setting cleaning cycle beforeprinting gives a uniform start every running. Cleaning cycle is veryimport for some high viscosity, because setting cleaning cycle duringprinting prevents the ink from clogging. And Setting cleaning cycleafter printing help you maintenance nozzles, as shown in FIG. 16.

2.2.4 Waveform

The DDM software has a standard 4 steps waveform which is good fornormal ink (viscosity: 11.12 centipoises; surface tension: 28.32dynes/cm). The 4 steps include start, phase 1, phase 2, and phase 3. Thebasic idea for those 4 steps is use a bias voltage to control piezo tosuck a drop of ink and jet it with a controlled velocity.

2.2.4.1 Waveform Start

At standby point, the voltage of nozzle sets to 40% level and holds itfor 1 μs. Under this condition, the channel which is piezo basicslightly deflected and sucks some ink from cartridge starting to eject,as shown in FIG. 17.

2.2.4.2 Waveform Phase 1

At the phase 1, set the voltage level to 0 and hold it for 3.584 μs. Thevoltage brings the piezo back to a neutral straight position withchamber at its maximum volume. In this phase, the fluid is filled intochamber. From the FIG. 18, it shows meniscus at the nozzle edge.

2.2.4.3 Waveform Phase 2

The phase 2 is firing pulse, as shown in FIG. 19. The steepness of theslope provides the energy for initial ejection and it is followed by ahold period. On the hold period, the voltage increases to 100% level andholds it for 3.712 μs. A this point, the chamber starts to jet a drop ofink. According to the hold time and voltage volume, the velocity of adrop can be calculated.

2.2.4.4 Waveform Phase 3

The last phase of the waveform is return to standby, as shown in FIG.20. First, the voltage level decreases to 70% and hold it for 3.392 μsthat is designed for prevent the printed head from sucking air back in.Second, the voltage level brings to 40% level and chamber back to thestandby position.

2.3 Conclusion

Those parameters what have be mentioned above play an important roleduring printing. They should be checked every printing running accordingto the ink material of substrate. Also ink condition has been slightchanged according to room temperature and humidity level.

3 SILK INKS

There are two kinds of ink including solvent based ink and water basedink. Solvent based ink usually contains some poisonous chemicalmaterial. And silk is processed in an all water-based, room temperature,neutral pH environment, is mechanically stable, edible, biocompatible,and implantable in the human body. Given the favorable materialproperties, the use of silk-based inks can be important for a variety ofcontrolled chemical and biological material fabrication on the micro-and nano. scale.

3.1 Water Based Inks

The majority of inkjet printers use water based inks. While everymanufacturer has its own ink formulations with specific chemicalconstituents, the main components in water based inks are similar. Thetable below describes the components that comprise water-based ink.

TABLE 6 Components in water based ink [20] Component FunctionConcentration, % water Aqueous carrier medium 60-90 Water solublesolvent Humectants, viscosity  5-30 control Dye or pigment Providescolor  1-10 Surfactant Wetting, penetrating 0.1-10  Buffer Controls thepH of ink 0.1-0.5 Other additives Chelating agent, defoamer <13.1.1 Humectants

The role of the humectants is to preserve the water content in ink, sothat it does not clog and dry out print head nozzles. People usually useglycerol, ethylene glycol as the humectants in the water based ink.

3.1.2 Surfactant

Surface tension is the main parameter for water based ink, becausedistilled water has a very high surface tension (70 dynes/cm). But theideal value of surface tension for inkjet printer to print is 28-44dynes/cm. A high surface tension ink will not wet the nozzle, so thatthe ink will not travel through the nozzle. Under this condition, theink will clop the nozzle. Furthermore, high surface tension will causethe ink not wet the substrate and result in uneven patterns on thesubstrate. But the low surface tension ink also causes some problem. Lowsurface tension induces fluid leaking from the nozzles. Surface tensionof the liquid drives the fluid into a spherical shape in order to reachminimum surface area, minimum energy state. The higher the surfacetension, the faster the fluid changes to a sphere. Adding just 1.34% ofa surfactant, P103 (BASF, USA) decreases the surface tension of waterfrom 69.5 dynes/cm to 33 dynes/cm at 20° C. [21]. Increasing thesurfactant to 5% has no effect on surface tension; the measured valuewas still 33 dynes/cm [21].

3.1.3 Viscosity Adjuster

Viscosity is a physical property that is often manipulated to makejet-able fluids. Viscosity is the reciprocal of fluidity and indicatesresistance to flow. The flow speed decreases as viscosity increases.Temperature increases fluid flow in Newtonian fluids. When a force isapplied to a volume of material, then displacement (deformation; i.e.flow) occurs. In ambient pressure, low temperatures push fluids towardstheir ordered state, and as fluids become more ordered, they alsoincrease in viscosity. On the other hand, heat can be applied todecrease apparent viscosity (increase fluidity), and the DMP ink jetprint head contains a heater thereby increasing the jettability windowfor viscous fluids. The ideal viscosity for inkjet printer to print is11 centpoises and the viscosity of water at room temperature is 1centipoises. Polyvinyl alcohol and glycerol add to water based ink asthe viscosity to increase the viscosity. Viscosity adjuster keeps ink atproper thickness so that it can be jetted smoothly and stabile.

3.1.4 Buffer

The role of the buffer is to maintain the PH level in the ink.

3.2 Fluid Requirement for Dimatix Materials Printer

Some ink physical characteristics to achieve optimum performance are[19]:

-   -   Viscosity—10-12 centipoises at jetting temperature    -   Surface Tension—28-44 dynes/cm at jetting temperature    -   Low Volatility—Boiling points higher than 100° C. are preferred    -   Density—Specific gravity greater than 1 is beneficial    -   Degassing—Additionally the fluid may need to be degassed to        remove any dissolve gas which inhibits jetting. Typical        degassing can be done with a vacuum (A negative pressure of 2        psi for 1-2 hours maybe sufficient or up to only 50 mbar),    -   Filtration—If particle size allows, it is recommended to filter        all fluids to 0.2 μm.    -   Acidity or Alkalinity—A pH . . . value between 4 and 9 is        recommended.

Note: The parameter of viscosity and surface tension is a pair ofcombine parameter. Fluid with surface tension higher than 44 combineswith viscosity lower than 10 also works for DMP 2800 printer bymodifying the waveform.

3.3 Silk Fibroin Ink

In order to go through the 0.2 μm filter, we developed a new method tomake a low molecular weight silk solution which can easy go through 0.2μm filter.

3.3.1 Method for Manufacturing High Temperature, High Pressure Silk

Example: for ˜40 mL of silk solution with a concentration of ˜6.25%(wt/vol), if more volumes are needed, the materials can be scaledappropriately.

-   1) Cut Bombyx mori silk cocoons (10 gram) into half-dime-sized    pieces and dispose of silkworms;-   2) Measure 8.48 gram of sodium carbonate and add it into 4 liter of    water in a 5 liter glass beaker (to prepare a 0.02 M solution);-   3) Put the beaker into an autoclave and set the autoclave to run at    121 degree C. under the pressure of 16 psi for 120 minutes;-   4) Remove the silk fibroin with a strainer and cool it by rinsing in    ultrapure cold water for 20 minutes and repeat twice for a total of    three rinses;-   5) After the third rinse, remove the silk fibroin and squeeze the    water;-   6) Spread the squeezed silk fibroin, spread it out and let it dry in    a fume hood for 12 hours, which results in silk fibroin weighing    slightly over 2.5 gram;-   7) Dissolve 2.5 gram of silk fibroin into 10 mL of 9.3 M lithium    bromide;-   8) The silk fibroin should dissolve completely in a few minutes upon    stirring;-   9) Insert 10 mL of the silk-LiBr solution into a pre-wet 3-12-mL    dialysis cassette and dialyze against 1 liter of ultrapure water for    48 hours (change the water every 6 hours);-   10) Remove silk from the cassette;-   11) Place the silk solution in a centrifuge and spin at 9,000 r.p.m.    at 2 degree C. for 60 minutes, and store the centrifuged silk    solution (˜40 mL of silk solution with a concentration of ˜6.25%) in    a refrigerator at 4 degree C.    3.3.2 Silk Ink Preparation

One important property Silk solution is that silk is easy getting Psheet when it is mixed with surfactant and viscosity adjuster. So, wejust add surfactant, like Tween 20, to silk solution. To increase theviscosity, adjust the waveform to compensate the viscosity.

Example: for ˜2 mL of silk fibroin protein ink, if more volumes areneeded, the materials can be scaled appropriately.

Mix the silk solution with surfactant (for example, Tween 20 fromSigma-Aldrich Co.) and water in a volume ratio of 17:2:1 (i.e. 1700 μLof ˜6.25% silk fibroin solution, 200 μL of Tween 20 and 100 μL ofwater);

Note that the ratio of the mixture is optimized for Tween 20 and otherbiological or chemical surfactant (for example, glycol, ether, and etc.)can be also used with modifications of the mixture ratio. Surfacetreatment of the printing nozzle(s) can also improve the formation ofsilk ink drops.

3.3.3 Surface Tension Measurement

Here are two tables for surface tension measurement of pure hightemperature silk solution and it mixed with surfactant.

TABLE 7 Surface Tension of Pure high temperature Silk Solution No. TimeGamma Opt Messages Beta R0 Area Volume Theta Height Width 1 0.0 46.830.261 1.118 19.67 8.20 109.48 3.014 2.350 2 2 0.1 46.81 0.261 1.11819.67 8.20 109.45 3.015 2.349 2 3 0.2 46.80 0.262 1.119 19.68 8.20109.36 3.016 2.350 2 4 0.3 46.80 0.262 1.119 19.68 8.20 109.29 3.0182.350 2 5 0.4 46.78 0.262 1.119 19.68 8.21 109.23 3.018 2.350 2 6 0.446.79 0.262 1.119 19.69 8.21 109.16 3.019 2.350 2 7 0.6 46.78 0.2621.119 19.69 8.21 109.09 3.020 2.351 2 8 0.6 46.76 0.262 1.119 19.70 8.21109.03 3.021 2.351 2 9 0.8 46.77 0.262 1.119 19.70 8.21 108.93 3.0222.351 2 10 0.8 46.75 0.262 1.119 19.71 8.22 108.84 3.023 2.351 2 11 0.946.78 0.262 1.119 19.71 8.22 108.92 3.023 2.351 2 12 1.0 46.75 0.2621.119 19.71 8.22 108.79 3.024 2.351 2 13 1.1 46.74 0.262 1.119 19.718.22 108.76 3.025 2.351 2 14 1.3 46.71 0.262 1.119 19.71 8.22 108.663.026 2.351 2 15 1.3 46.69 0.262 1.119 19.72 8.22 108.58 3.027 2.351 216 1.4 46.67 0.262 1.119 19.72 8.22 108.54 3.028 2.351 2 17 1.6 46.670.262 1.119 19.72 8.22 108.52 3.028 2.351 2 18 1.6 46.65 0.262 1.11819.72 8.22 108.44 3.029 2.351 2 19 1.8 46.66 0.262 1.119 19.72 8.22108.45 3.029 2.351 2 20 1.9 46.63 0.262 1.119 19.72 8.22 108.33 3.0312.351 2 21 2.0 46.65 0.262 1.119 19.73 8.22 108.35 3.030 2.351 2 22 2.146.62 0.263 1.119 19.73 8.22 108.28 3.031 2.351 2 23 2.1 46.64 0.2621.119 19.73 8.22 108.19 3.032 2.351 2 24 2.3 46.54 0.263 1.118 19.738.22 108.02 3.034 2.350 2 25 2.3 46.61 0.263 1.119 19.73 8.22 108.133.032 2.351 2 26 2.4 46.58 0.263 1.118 19.73 8.22 108.06 3.034 2.350 227 2.5 46.59 0.263 1.118 19.74 8.22 108.01 3.034 2.351 2 28 2.7 46.570.263 1.118 19.74 8.23 107.98 3.035 2.350 2 29 2.8 46.55 0.263 1.11819.74 8.22 107.91 3.036 2.350 2 30 2.8 46.54 0.263 1.118 19.74 8.22107.86 3.036 2.350 2 31 2.9 46.53 0.263 1.118 19.74 8.23 107.86 3.0362.351 2 32 3.1 46.56 0.263 1.118 19.74 8.23 107.84 3.037 2.350 2 33 3.246.51 0.263 1.118 19.75 8.23 107.76 3.038 2.351 2 34 3.3 46.56 0.2631.119 19.75 8.23 107.82 3.037 2.351 2 35 3.3 46.52 0.263 1.118 19.758.23 107.68 3.039 2.351 2 36 3.5 46.53 0.263 1.118 19.75 8.23 107.713.039 2.351 2 37 3.5 46.51 0.263 1.118 19.75 8.23 107.66 3.039 2.351 238 3.7 46.49 0.263 1.118 19.75 8.23 107.58 3.040 2.351 2 39 3.7 46.510.263 1.118 19.75 8.23 107.63 3.040 2.351 2 40 3.8 46.49 0.263 1.11819.76 8.23 107.48 3.041 2.350 2 41 4.0 46.49 0.263 1.118 19.76 8.23107.53 3.041 2.351 2 42 4.0 46.48 0.263 1.118 19.76 8.23 107.50 3.0412.351 2 43 4.1 46.48 0.263 1.118 19.76 8.23 107.45 3.041 2.350 2 44 4.246.48 0.263 1.118 19.76 8.23 107.43 3.042 2.351 2 45 4.3 46.48 0.2631.118 19.76 8.23 107.42 3.042 2.350 2 46 4.5 46.45 0.263 1.118 19.768.23 107.28 3.043 2.350 2 47 4.5 46.43 0.263 1.118 19.76 8.23 107.303.043 2.350 2 48 4.7 46.45 0.263 1.118 19.76 8.23 107.36 3.042 2.350 249 4.7 46.44 0.263 1.118 19.76 8.23 107.30 3.043 2.350 2 50 4.8 46.430.263 1.118 19.76 8.23 107.14 3.045 2.350 2 Mean: 46.61 0.263 1.11819.73 8.22 108.19 3.032 2.350 Stand.dev. 0.02 0.000 0.000 0.00 0.00 0.090.001 0.000

TABLE 8 Surface Tension of high temperature Silk Solution Mix with 10%surfactant No. Opt Time Gamma Beta R0 Area Volume Theta Height Width 10.0 39.83 0.275 1.059 17.45 7.04 117.75 2.737 2.231 2 2 0.4 39.73 0.2761.059 17.53 7.05 116.61 2.759 2.232 2 3 0.9 39.60 0.277 1.058 17.59 7.07115.46 2.779 2.230 2 4 1.4 0.00 0.000 0.000 0.00 0.00 0.00 0.000 0.000 0Sides are too differ 5 1.9 39.33 0.278 1.057 17.68 7.08 113.17 2.8162.229 2 6 2.4 39.27 0.278 1.057 17.73 7.09 112.11 2.832 2.229 2 7 3.039.07 0.279 1.056 17.74 7.08 110.96 2.846 2.227 2 8 3.4 39.16 0.2791.057 17.80 7.10 110.03 2.862 2.228 2 9 3.9 39.11 0.279 1.056 17.84 7.11108.96 2.877 2.228 2 10 4.4 39.08 0.279 1.056 17.87 7.12 107.81 2.8912.228 2 11 4.9 0.00 0.000 0.000 0.00 0.00 0.00 0.000 0.000 0 Error inprofile 12 5.4 38.98 0.280 1.056 17.94 7.13 105.43 2.921 2.227 2 13 5.938.96 0.280 1.055 17.98 7.13 103.73 2.941 2.226 2 14 6.4 38.94 0.2801.055 18.03 7.13 101.75 2.964 2.225 2 15 7.0 38.93 0.280 1.055 18.077.14 99.67 2.987 2.225 2 16 7.5 38.92 0.280 1.054 18.12 7.14 97.29 3.0122.223 2 17 8.0 38.94 0.279 1.054 18.18 7.14 94.20 3.044 2.223 2 18 8.40.00 0.000 0.000 0.00 0.00 0.00 0.000 0.000 0 Error in profile 19 8.90.00 0.000 0.000 0.00 0.00 0.00 0.000 0.000 0 Error in profile 20 9.51.26 4.182 0.733 1.53 0.18 32.45 0.276 1.288 7 Mean: 36.82 0.523 1.03616.82 6.67 102.96 2.722 2.169 Stand.dev. 2.37 0.244 0.020 1.02 0.43 5.010.165 0.059

Compare the surface tension of high temperature silk solution with 60minutes boiled silk from Table 9 and Table 10. Apparently, the surfacetension of the high temperature silk solution is approach to 60 minutesboiled silk, but it has a higher viscosity.

TABLE 9 Surface Tension of Pure 60 Minutes Boiled Silk Solution No. TimeGamma Opt Messages Beta R0 Area Volume Theta Width Height 1 0.0 45.220.249 1.073 17.64 7.06 118.23 2.767 2.248 2 2 0.1 44.98 0.251 1.07417.71 7.09 117.52 2.780 2.251 2 3 0.2 44.77 0.252 1.074 17.77 7.12116.80 2.792 2.252 2 4 0.3 44.65 0.253 1.075 17.84 7.15 115.93 2.8062.254 2 5 0.4 44.42 0.254 1.075 17.88 7.17 115.21 2.816 2.255 2 6 0.444.39 0.255 1.075 17.91 7.18 114.99 2.821 2.256 2 7 0.6 44.47 0.2551.076 17.92 7.19 115.12 2.821 2.258 2 8 0.7 44.38 0.255 1.076 17.94 7.20114.82 2.826 2.258 2 9 0.8 44.24 0.256 1.076 17.97 7.21 114.42 2.8322.258 2 10 0.9 44.24 0.256 1.076 17.98 7.22 114.30 2.834 2.259 2 11 0.944.25 0.256 1.076 17.99 7.23 114.28 2.835 2.259 2 12 1.1 44.15 0.2571.076 18.01 7.23 113.97 2.840 2.259 2 13 1.1 44.09 0.257 1.076 18.027.24 113.75 2.843 2.259 2 14 1.3 44.07 0.257 1.076 18.03 7.24 113.632.845 2.260 2 15 1.3 44.05 0.257 1.077 18.04 7.25 113.53 2.847 2.261 216 1.4 44.00 0.258 1.077 18.05 7.25 113.41 2.849 2.260 2 17 1.6 43.960.258 1.077 18.06 7.25 113.22 2.851 2.261 2 18 1.6 43.96 0.258 1.07718.07 7.26 113.19 2.852 2.261 2 19 1.7 43.93 0.258 1.077 18.07 7.26113.10 2.854 2.261 2 20 1.8 43.89 0.258 1.077 18.08 7.26 112.96 2.8562.261 2 21 1.9 43.86 0.259 1.077 18.09 7.27 112.82 2.857 2.261 2 22 2.043.84 0.259 1.077 18.09 7.27 112.79 2.858 2.261 2 23 2.1 43.83 0.2591.077 18.10 7.27 112.67 2.860 2.262 2 24 2.2 43.81 0.259 1.077 18.107.27 112.63 2.861 2.261 2 25 2.3 43.78 0.259 1.077 18.11 7.28 112.522.862 2.261 2 26 2.4 43.79 0.259 1.077 18.11 7.28 112.47 2.863 2.262 227 2.6 43.76 0.259 1.077 18.12 7.28 112.41 2.864 2.261 2 28 2.6 43.740.260 1.077 18.12 7.29 112.33 2.866 2.262 2 29 2.8 43.72 0.260 1.07718.13 7.29 112.22 2.867 2.262 2 30 2.8 43.71 0.260 1.077 18.13 7.29112.20 2.868 2.262 2 31 2.9 43.70 0.260 1.077 18.14 7.29 112.14 2.8692.262 2 32 3.1 43.68 0.260 1.077 18.14 7.29 112.10 2.869 2.262 2 33 3.143.67 0.260 1.077 18.15 7.30 112.00 2.870 2.263 2 34 3.3 43.64 0.2601.077 18.15 7.30 111.94 2.871 2.262 2 35 3.3 43.65 0.260 1.077 18.157.30 111.92 2.872 2.262 2 36 3.4 43.64 0.260 1.077 18.16 7.30 111.842.873 2.263 2 37 3.5 43.64 0.260 1.077 18.16 7.30 111.78 2.873 2.263 238 3.6 43.62 0.260 1.077 18.16 7.30 111.78 2.874 2.263 2 39 3.8 43.610.260 1.077 18.17 7.30 111.73 2.875 2.263 2 40 3.8 43.59 0.260 1.07718.17 7.31 111.68 2.876 2.263 2 41 4.0 43.58 0.261 1.077 18.17 7.31111.62 2.876 2.263 2 42 4.0 43.57 0.261 1.077 18.17 7.31 111.57 2.8772.264 2 43 4.1 43.56 0.261 1.077 18.18 7.31 111.54 2.878 2.263 2 44 4.343.54 0.261 1.077 18.18 7.31 111.49 2.879 2.263 2 45 4.3 43.55 0.2611.077 18.19 7.31 111.44 2.879 2.264 2 46 4.4 43.52 0.261 1.077 18.197.31 111.37 2.880 2.263 2 47 4.5 43.52 0.261 1.077 18.19 7.31 111.352.880 2.264 2 48 4.7 43.52 0.261 1.077 18.19 7.32 111.32 2.881 2.264 249 4.8 43.50 0.261 1.077 18.19 7.32 111.30 2.881 2.263 2 50 4.8 43.490.261 1.077 18.20 7.32 111.25 2.882 2.264 2 Mean: 43.91 0.258 1.07718.07 7.26 113.01 2.854 2.261 Stand.dev. 0.06 0.000 0.000 0.02 0.01 0.240.004 0.000

TABLE 10 Surface Tension of Pure 60 Minutes Boiled Silk Solution Mixwith 10% surfactant No. Time Gamma Opt Messages Beta R0 Area VolumeTheta Height Width 1 0.0 37.44 0.278 1.031 17.14 6.63 104.04 2.868 2.1752 2 0.4 37.40 0.278 1.031 17.15 6.64 103.76 2.871 2.175 2 3 0.9 37.380.278 1.031 17.16 6.64 103.55 2.874 2.175 2 4 1.4 37.35 0.279 1.03117.17 6.65 103.26 2.878 2.175 2 5 1.9 37.34 0.279 1.031 17.18 6.65103.15 2.880 2.175 2 6 2.4 37.32 0.279 1.031 17.18 6.65 102.95 2.8822.175 2 7 2.9 37.32 0.279 1.031 17.19 6.65 102.84 2.884 2.175 2 8 3.537.31 0.279 1.032 17.20 6.66 102.65 2.887 2.176 2 9 4.0 37.28 0.2791.032 17.21 6.66 102.48 2.889 2.176 2 10 4.5 37.29 0.279 1.032 17.216.66 102.32 2.891 2.176 2 11 5.0 37.26 0.279 1.032 17.22 6.66 102.192.892 2.176 2 12 5.5 37.27 0.279 1.032 17.22 6.66 102.03 2.894 2.176 213 6.0 37.26 0.279 1.032 17.23 6.67 101.93 2.896 2.176 2 14 6.5 37.260.279 1.032 17.23 6.67 101.88 2.897 2.177 2 15 7.0 37.24 0.280 1.03217.24 6.67 101.73 2.899 2.176 2 16 7.5 37.23 0.280 1.032 17.24 6.67101.62 2.900 2.176 2 17 8.0 37.25 0.280 1.032 17.25 6.67 101.62 2.9002.177 2 18 8.4 37.23 0.280 1.032 17.25 6.67 101.48 2.902 2.177 2 19 9.037.22 0.280 1.032 17.25 6.67 101.36 2.903 2.176 2 20 9.4 37.22 0.2801.032 17.26 6.68 101.30 2.904 2.177 2 Mean: 37.29 0.279 1.032 17.21 6.66102.41 2.890 2.176 Stand.dev. 0.01 0.000 0.000 0.01 0.00 0.19 0.0020.0003.3.4 Printable Substrates for Silk Inks

The printable substrates using silk fibroin inks are limitless, simplydepending on the available inkjet printers. The printable substratesinclude, but not limited to, the followings:

-   -   Paper    -   Glass and other insulators    -   Silicon and other semiconductors    -   Metals    -   Cloth textiles    -   Plastics

4 INKJET PRINTED SILK PATTERNS

We use the DMP-22800 printer to print some silk patterns, like dots,signal line, and 2D patterns, on both hydrophilic and hydrophobicsubstrates. The resolution of printing affects by viscosity and surfacetension of the silk ink. Also the resolution of pattern depends onroughness of substrate and nozzle size. The DMP-2800 printer provides a10 μl size nozzle to make patterns, so one drop size is around 25 μm andthe width of a line is around 40 um on the hydrophilic substrates. Asignal layer line will give the interface between dots, and a 2D patternpresents interface between lines.

4.1 Parameter Setting for Silk Ink

4.1.1 Voltage

The voltage is function of drop size and drop velocity. So voltagesetting depend on height level what you want the nozzle above yoursubstrate and the drop size your want to print. But voltage level below15 V, the silk ink will not come out due to the surface tension of silkink. High Voltage setting gives you more volume of the drop. FIG. 21shows silk lines on silicon wafer under 15 v, 20V and 25 v voltageprinting, and the width of silk lines are 65 μm, 100 μm, 110 μm.Obviously, High voltage printing gives more width lines due toincreasing the drop volume.

4.1.2 Waveform

After adding the surfactant to silk solution, the surface tension of theink is about 36 dynes/cm. So we use the waveform which is developed bythe Dimatix Company.

4.1.3 Cleaning Cycle

Before printing, we need a purging process that applies air pressure tooutside of fluid bag to force fluid through entire fluid path and outall nozzles at nozzle, as shown in FIG. 22. After purging process, itforce air in the chamber out the nozzles, and make sure ink wet thenozzle to start printing.

During printing, blotting process is required to absorbent silk ink inclose to nozzle plate. After blotting process, the excess silk ink whichcauses misdirected firing will be removed.

After printing, spitting process provides protect nozzle from clogging.Spitting designed to ejecting some drops ink from the chamber. It letsthe fresh silk drops reach to the meniscus to replace the old one.

4.1.4 Nozzle Number

The number of nozzle also affects the printing patterns. FIG. 23 shows asilk line on the acrylic with 25 v and one nozzle printing, and it gives40 μm width silk line. FIG. 24 is silk line with 240 widths still under25 v printing. However, the width of the line become much more widththan FIG. 23, because it printing by 7 nozzles.

4.1.5 Silk Drops from Nozzle

FIG. 25 is the drops from 10 μL nozzles, and the voltage value set 23V,jetting frequency is 5 KHz. The uniform drops from nozzle performancestable. There are no misdirected nozzles which mean that silk solutionjetting smooth without bubbles under the high frequency oscillatesystem. All of the sixteen nozzles work well last for 8 hours whichmeans the high temperature silk will not clog the 20 μm diameter nozzle.

4.2 Various Silk Patterns Using Directly Inkjet Printing Technique

4.2.1 Dots

FIG. 26 and FIG. 27 show silk dots printed on silicon wafer and acrylic,respectively. We use 1 nozzle and 1 layer printing, the voltage value is15 v and jetting frequency is 1 KHz. The size of dots is 40 μm onsilicon wafer and 30 μm on acrylic.

4.2.2 Lines

4.2.2.1 Multiple Layers Printing

FIG. 28 shows silk lines which are printed with one nozzle and 15 v onthe silicon wafer. And FIG. 29 shows the SEM picture of those one layerprinting. One layer printing is clear without any interface betweendrops. Comparing one layer printing with three layers printing, onelayer patterns are more uniform and the edge of line is cleaner. A roughedge shows on three layers printing (FIG. 30), because the upper layerfluid causes capillary instability when the upper layers silk areprinted. From the FIG. 31, it indicates the first line is width thanother 4 line, because the alignment of first line is not as good asother 4 lines. FIG. 32 shows serious capillary instability in a twentylayers pattern, so multiply layer printing is just suitable for lowresolution patterns.

4.2.2.2 Cross Lines Printing

The method for printing multiple layers lines and cross lines isdifferent. For multiple layers lines, the substrate is fixed duringprinting and the direction of printing among multiply layers lines issome. For the cross lines printing, the substrate is routed by 90 degreeC. after first layer printing, and then do the second layer printing. Sothe direction of the two layers is different. The FIGS. 33-35 indicatescapillary instability between two layers, and the edge of pattern showsa clean gradual capillary instability process.

4.2.3 Two Dimension Printing

One layer square pattern shows interface between lines. From FIG. 36,there is less than 1 μm width overlap between two lines. After applyinglaser point to the pattern, a diffraction grating patterns show on thewall due to the 1 μm overlap, as shown in FIG. 37. However, the overlappart of the pattern is disappeared after printing second layer pattern.So, the multiple layers give a smooth finish pattern (FIGS. 38 and 39).

4.2.4 Silk Patterns after Alcohol Annealing Treatments

Silk film is easily dissolved in the water. However it will not dissolveafter alcohol annealing due to the formation of β sheet. We try to useprinter to make a β sheet pattern. So we put the printing pattern to doa 2 hours vacuum annealing. It turns out the patterns tend to spreadout, as shown in FIG. 40.

4.3 Thickness of Silk Pattern

Silk provides a biologically favorable environment allowing them toentrain various biological and chemical dopants and maintain theirfunctionality. Mixing Different chemical solution with silk solutiongives different viscosity and surface tension which are affect thicknessof pattern. Obviously, the number of printing layer is another importantelement affects the thickness of pattern. Preparing three kinds of silksolution include food color silk, high refractive index silk and puresilk, and then print them with some number nozzles. FIGS. 41-43 showsthe thickness of patterns are increased by the number printing layer.The thinnest pattern is less than 100 nm created by one layer food colorsilk pattern. According to FIG. 44 the thickest pattern is pure silkpattern due to highest percentage silk in the solution.

4.4 Silk Patterns on Various Substrates

The printable substrates for silk ink include paper, glass, silicon,metals, cloth textiles and plastics. Those substrates can be divided twogroups which are hydrophobic substrate and hydrophilic substrate. Thedrop size on hydrophobic substrate is sight smaller due to high surfaceenergy. The width of silk lines from FIG. 28 is similar with the silklines from FIG. 45. However, the two patterns are supplied by differentvoltage. Silk patterns on silicon have slight large voltage value.

5 DIRECTLY PRINTING OF FUNCTIONAL SILK DEVICES USING DOPED SILK SOLUTIONAS THE INK

Silk fibroin has proved to be an effective material and matrix that canmaintain the functionalities of dopants. Therefore, choosing theappropriate dopants (including both physical dopants—e.g. metallicnanoparticles, laser dyes, quantum dots and etc.—and biochemicaldopants, e.g. cells, enzymes, bacterium and etc.) and mixing them intosilk fibroin solution as the ink is a promising way to directly printingof functional devices using Dimatix DMP 2800 printer. In the followingsection, a series of functional silk devices (with different dopants)will be described as the proof of principle demonstrations.

5.1 Inkjet Printing of Gold Nanoparticle Doped Silk Patterns

As mentioned above, silk provides a biologically favorable environmentallowing them to entrain various biological and chemical dopants andmaintain their functionality. Proteins and enzymes haven been previouslydoped into various silk material formats, especially silk films.Recently, we have demonstrated gold nanoparticles doped silk films thatresonantly absorb incident light and convert it to heat, which can bepotentially used as a biocompatible thermal therapy for in vivo medicalapplications such as tumor and bacterial killing.

The preparation of gold nanoparticle silk ink consist of the productionof the print grade silk fibroin solution and synthesis of goldnanoparticles, followed by a simple mixing of the two solution with acertain ratio that is determined by applications. Briefly, pre-cutBombyx mori cocoon pieces are boiled in a 0.02 M Na₂CO₃ solution for 2hours to remove sericin and boiled silk fibers are dried overnight andthen are siddolved in a 9.3 M LiBr at 60 degree C. for 4 hours. Thelithium bromide salt is then removed from the silk solution through awater-based dialysis process. The gold nanoparticle solution is preparedby adding 20 mL 1% Na₃C₆H₅O₇ into 200 mL boiled 1.0 mM HAuCl₄, followedby continuously heating for 10 minutes until the solution has turneddeep red. Then the gold nanoparticle solution is carefully added intothe silk solution with gentle agitation for uniform dispersion and isready for printing after being filtered against 0.2 micron filter.

Table 1 gives the main parameters for printing and the printing resultis showed in FIG. 47.

The printed Au—NPs doped silk device showed enhanced plasmaticabsorption of green light (FIG. 48), resulting in a temperature increaseof ˜15 degree s with an irradiance of ˜0.25 W/cm². The heating effectscould be further improved and optimized by adjusting the Au-NPsconcentration and layers of the printed structures, which could bepotentially used for light-mediated patterned heating treatments.

TABLE 1 Printing Permanents for Gold Nanoparticle Silk Ink Voltage 25 vNozzle Number  4 Drop Spacing 25 μm Printing Layer  5 Firing Frequency 2 KHz5.2 Inkjet Printing of Enzyme Doped Silk Patterns

In addition to print gold nanoparticle doped silk, it is also possibleto directly print enzyme—doped silk for biomedical applications such asenzyme-linked immuosorbent assay test (i.e. ELISA). ELISA is a widelyused test to identify certain substance using antibodies and thecolorimetric change as the sensing/diagnostic mechanism. Usually, theenzymes used in ELISA tests need to be stored at low temperature formaintaining the bioactivities. It has been proved that silk can help tomaintain the functionalities of the doped enzyme at room temperatureswithout fridge-storage. Therefore, directly printing of enzyme dopedsilk patterns (in a precise way) holds great opportunities in such asrapid and low volume screening test, food allergens, and toxicologyapplications, as shown in FIG. 49.

5.3 Inkjet Printing of Antibiotics Doped Silk Patterns

The use of antibiotics is important for effective infectious diseasecontainment and curing. However, for most, if not all, of currentantibiotics need to be maintained within a specific refrigerationtemperature range due to their temperature sensitivity. Silk fibroin hasbeen proven to be a biologically friendly protein polymer. Recently,researchers found that silk was capable of stabilizing labileantibiotics (in the form of films) even at temperatures up to 60 degreeC. over more than 6 months. We have been working on exploration of thepossibilities of directly inkjet printing of antibiotics doped silk bymixing penicillin solution of various concentration levels with purifiedsilk solution prepared as previously described. Compared to antibioticsdoped silk films, directly printing of antibiotics doped silk has theadvantages of precise control of the antibiotics distribution andpotential multilayer and multi-drugs printing that may benefit moresophisticated cases where fine control and micro-manipulation of theantibiotic drug are needed.

To obtain a clear pattern on bacterial growth area, first we use methodone try to print pattern before bacterial growth. The result shows thattwo clean square without any clean pattern in the Petri dish after 5hours incubate, as shown in FIG. 50.

Method one:

-   -   1) Culture 50 ul bacterial on agar    -   2) Print 1 layers an arrow and “tufts” on bacterial, the drop        gap 50 um    -   3) Take 5 hours culture in 37 degree C. incubator

To improve the method, the pattern is printed after bacterial overnightgrowth (Method two). There is an arrow in the in the Petri dish after 9hours incubate (FIG. 51).

Method two:

-   -   1) Culture bacterial on agar    -   2) Take overnight culture in 37 degree C. incubator    -   3) Print 2 layers an arrow and 25 um drop gap on bacterial    -   4) Take 9 hours culture in 37 degree C. incubator        5.4 Inkjet Printing of Colored Silk Patterns

In addition to biomedical applications (especially for implantableones), silk has been used to construct edible food sensors [22] as agreen and edible material that is extracted and purified fromdomesticated silkworm cocoons. Plain silk solution (i.e. non-doped silksolution) is a water-like highly transparent protein solution that iscolorless.

Food coloring, alternatively called color additive, imparts color whenadded to food or drink, and is used widely both in commercial foodproduction and in domestic cooking. We mix commercially available fooddyes (considered as safe) with silk solution to make colored silk inksand try to directly inkjet print. And we try to print patterns ontextile silk which carries a basic color (light yellow).

To get clear pattern on textile silk, it needs multiple layers printing,because the color of textile silk darker than a blank paper. After 7layers printing, the pattern is clear and beautiful, as shown in FIG.52.

The color silk patterns remain its original pattern after 2 hours vacuumannealing. Also it is survival after dry cleaning process, as shown inFIG. 53.

Multiple colors silk printing needs the alignment process, because theprinter loads one cartridge with one color at one once, as shown in FIG.54. There are four steps alignments including multiple layers alignment,cartridge, voltage alignment and voltage alignment.

Multilayer alignment: one color for each layer printing;

Cartridge alignment: set drop offset before every layer printing;

Voltage alignment: different color inks have slight change in viscosity;

Nozzle alignment: using same nozzles for every layer printing (number ofnozzles determines line width).

6 CONCLUSION

We have successfully demonstrated direct printing of silk fibroinprotein based inks using a commercially available inkjet printer.Various types of silk inks have been prepared by choosing appropriatedopants and mixing with the purified silk fibroin solution, and printedsuccessfully. A set of operating parameters have been tried andoptimized for each individual silk ink (including gold nanoparticle silkink, enzyme doped silk ink, high refractive index silk ink andantibacterial silk ink) to improve the performance for specificapplications. Both single layer and multiple layers printing have beencarried out and a resolution of 25 microns has been achieved.

Future work of silk ink printing would be focused on improving the silkink properties—in terms of printing speed and resolution—to match toother inkjet printers in the marketing. More efforts should be put onimproving the repeatability of the printing process, including avoidingthe clogging within the nozzles when the silk ink gets dried, and theaccuracy of step motor movement and alignment.

Another priority will be further studies of the effects of the nozzlejetting process on the properties of the silk ink, especially on theformation of silk beta sheet.

REFERENCES

-   1. G. H. Altman, F. Diaz, C. Jakuba, T. Calabro, R. L. Horan, J.    Chen, H. Lu, J. Richmond, D. L. Kaplan, “Silk-based biomaterials”,    Biomaterials 24 (2003) 401-416.-   2. D. L. Kaplan, S. M> Mello, S. Arcidacono, S. Fossey, K.    Senecal, W. Muller, “Protein based materials”, BOston:    Birkhauser, (1998) p. 103-131.-   3. D. L. Kaplan, W. W. Adams, B. Farmer, C. Viney, “Silk: biology,    structure, properties and genetics.” in the book of “Silk polymers:    materials science and biotechnology”. ACS Symp Ser (1994); 544:2-16.-   4. D. L. Kaplan, S. Fossey, C. Viney, W. Muller, “Self-organization    (assembly) in biosynthesis of silk fibers—a hierarchical problem.”    in the book of “Hierarchically structured materials” Materials Res    Symp Proc (1992); 255:19-29.-   5. R. L. Moy, A. Lee, A. Zakla, “Commonly used suture materials in    skin surgery”, American Family Physician, (1992); 44:2123-8.-   6. C. M. Wen, S. T. Ye, L. X. Zhou, Y. Yu, “Silk-induced asthma in    children: a report of 64 cases”, Annals of Allergy, Asthma &    Immunology, (1990); 65: 375-8.-   7. E. Rossitch, D. E. Bullard, W. J. Oakes, “Delayed foreign-body    reaction to silk sutures in pediatric neurosurgical patients”,    Child's Nervous System, (1987); 3: 375-8.-   8. M. Dewair, X. Baur, K. Ziegler, “Use of immunoblot technique for    detection of human IgE and IgG antibodies to individual silk    proteins”, Journal of Allergy and Clinical Immunology, (1985); 76:    537-42.-   9. H. Sakabe, T. Miyamoto, Y. Noishiki, W. S. Ha, “In vivo blood    compatibilityof regenerated silk fibroin”, Gen-I Gakkaishi, (1989);    45: 487-90.-   10. M. Santin, A. Motta, G. Freddi, M. Cannas, “In vitro evaluation    of the inflammatory potential of the silk fibroin”, Journal of    Biomedical Materials Research, (1999); 46: 382-9.-   11. H. Peleg, U. N. Rao, L. J. Emrich, “A experimental comparison of    suture materials for tracheal and bronchial anastomoses”, The    Journal of Thoracic and Cardiovascular Surgery, (1986); 34: 384-8.-   12. H. J. Jin, J. Chen, V. Karageorgiou, G. H. Altman, D. L. Kaplan,    “Human bone marrow stromal cell responses on electrospun silk    fibroin mats”, Biomaterials, (2004); 25 (6):1039 . . . 1047.-   13. Y. Cao, B. Wang, “Biodegradation of silk biomaterials”,    International Journalof Molecular Sciences, (2009); 10: 1514-24.-   14. C. Vepari, D. L. Kaplan, “Silk as a biomaterial”, (2007); 32:    991-1007.-   15. D. N. Rockwood, R. C. Preda, T. Yucel, X. Wang, M. L.    Lovett, D. L. Kaplan, “Materials fabrication from Bombyx mori silk    fibroin”, Nature Protocol, (2011); 6 (10): 1612-31.-   16. FUJIFILM Dimatix DMP 2800 printer user's manual.-   17. A. H. Rida, “Conductive inkjet printed antennas on flexible    low-cost paper-based substrates for RFID and WSN applications”,    Masters' thesis, Georgia Institute of Technology, May 2009.-   18. G. Shaker, S. Safavi-Naeini, N. Sangary, M. M. Tentzeris,    “Inkjet printing of ultrawideband (UWB) antennas on paper-based    substrates”, IEEE Antennas and Wireless Propagation Letters, Vol.    10, (2011); 111-114.-   19. B. Derby, “Bioprinting: inkjet printing proteins and hybrid    cell-containing materials and structures”, Journal of Materials    Chemistry, (2008), 18: 5717-21.-   20. http://www.imaging.org/resources/leinkjet/part4.cfm-   21. Jan Sumerel, unpublished observation-   22. H. Tao, M. A. Brenckle, M. Yang, J. Zhang, M. Liu, S. M.    Siebert, R. D. Averitt, M. S.

Mannoor, M. C. McAlpine, J. A. Rogers, D. L. Kaplan, F. G. Omenetto,“SIlk-based conformal, adhesive, edible food sensors”, AdvancedMaterials, (2012).

What is claimed is:
 1. A printable keratin ink comprising: an aqueouskeratin solution, substantially free of an organic solvent and having amolecular weight of about 3.5 kD to about 350 kD and a concentration ofkeratin in a range of about 0.1 (wt/vol) % to about 10 (wt/vol) %;wherein when it is printed to a substrate, a droplet volume of about 0.1pL to about 5 nL of the aqueous keratin solution is characterized by aviscosity of about 1 centipoise to about 20 centipoise when viscosity ismeasured at room temperature and a surface tension of about 15 dynes/cmto about 50 dynes/cm when measured at room temperature.
 2. The printablekeratin ink of claim 1, having a pH value between 5-9.
 3. The printablekeratin ink of claim 1, further comprising a viscosity-modifying agent,a surfactant, or combination thereof.
 4. The printable keratin ink ofclaim 3, wherein the viscosity-modifying agent is present at about 5-30wt % of the printable keratin ink composition.
 5. The printable keratinink of claim 3, wherein the surfactant is present at about 0.1-10 wt %of the printable keratin ink composition.
 6. The printable keratin inkof claim 1, further comprising an additive.
 7. The printable keratin inkof claim 1, further comprising a structural protein selected from thegroup consisting of: fibroins, actins, collagens, catenins, claudins,coilins, elastins, elaunins, extensins, fibrillins, lamins, laminins,tublins, viral structural proteins, zein proteins and any combinationsthereof.
 8. The printable keratin ink of claim 7, wherein the structuralprotein is a low molecular weight structural protein selected from thegroup consisting of: low molecular weight silk fibroins, low molecularweight actins, low molecular weight collagens, low molecular weightcatenins, low molecular weight claudins, low molecular weight coilins,low molecular weight elastins, low molecular weight elaunins, lowmolecular weight extensins, low molecular weight fibrillins, lowmolecular weight lamins, low molecular weight laminins, low molecularweight tublins, low molecular weight viral structural proteins, and anycombinations thereof.
 9. The printable keratin ink of claim 1, whereinthe printable keratin ink is substantially free of keratin having amolecular weight over 200 kDa.
 10. The printable keratin ink of claim 1,wherein the droplet unit printed to the substrate, has a gel,semi-solid, or solid form and a diameter measured at its smallestcross-section of about 0.1 μm to about 250 μm.
 11. The printable keratinink of claim 1, wherein the aqueous keratin solution is characterizedsuch that when it is printed to a substrate, a gel, a semi-sold, or asolid unit forms having a resolution about 5 dpi to about 20,000 dpi.12. A method for printing a structure, the method comprising steps of:providing the printable keratin ink of claim 1; depositing the printablekeratin ink through a nozzle in liquid droplets onto a substrate in apredetermined spatial pattern, wherein each liquid droplet has a volumeof about 0.1 pL to about 5 nL.
 13. The method of claim 12, wherein thedepositing step comprises a jetting velocity of about 7 m/sec to about 9m/sec.
 14. The method of claim 12, further comprising a step ofpiezoelectrically actuating the nozzel.
 15. The method of claim 12,wherein the nozzle has a diameter in the range of 10 and 50 μm.
 16. Themethod of claim 12, further comprising a step of applying at least oneof (a) heat and (b) ultraviolet irradiation to the aqueous keratin inkafter depositing the aqueous keratin ink onto the substrate.
 17. Themethod of claim 12, wherein the aqueous keratin ink further comprises adopant.
 18. The method of claim 17, wherein the dopant comprises ananoparticle.
 19. A printed array comprising: a substrate; and, aplurality of dot units, wherein the plurality of dot units is in a gelform, a semi-solid form or a solid form, and wherein dot units of theplurality of dot units were formed, jetted, printed and/or depositedfrom the printable keratin ink of claim 1 upon a substrate, such thatthey form in a predetermined spatial pattern on a surface of thesubstrate.
 20. The printed array of claim 19, wherein the printed arrayhas a resolution of between about 50-20,000 dpi.
 21. The printed arrayof claim 19, wherein the printed array forms substantially atwo-dimensional (2D) structure having a predetermined spatial pattern ofsubstantially even thickness.
 22. The printed array of claim 19, whereinthe printed array forms substantially a three-dimensional (3D) structurehaving a predetermined spatial pattern of varying thickness across thepredetermined spatial pattern.
 23. The printed array of claim 19,wherein the dot units of the plurality of dot units are about 0.1 μm toabout 250 μm in diameter.
 24. The printed array of claim 19, wherein thedot units of the plurality of dot units have a volume of about 0.1 pL toabout 5 nL.